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Trehalose 6-phosphate phosphataAQ1 seAQ2

s ofAQ3 Pseudomonas aeruginosa

Megan Cross1 Sonja Biberacherdagger1 Suk-Youl ParkDagger1 Siji Rajan Pasi Korhonensect Robin B Gassersect

Jeong-Sun Kim Mark J Coster and Andreas Hofmannsectk2

Griffith Institute for Drug Discovery Griffith University Nathan Queensland Australia daggerDepartment of Biology Friedrich-AlexanderUniversity Erlangen-Nuremberg Erlangen Germany DaggerPohang Accelerator Laboratory Pohang University of Science and TechnologyPohang Gyeongbuk South Korea sectDepartment of Veterinary Biosciences Melbourne Veterinary School The University of MelbourneParkville Victoria Australia Department of Chemistry Chonnam National University Gwangju South Korea and kQueensland TropicalHealth Alliance Smithfield Queensland Australia

ABSTRACT The opportunistic bacterium Pseudomonas aeruginosahas been recognized as an important pathogen ofclinical relevance and is a leading cause of hospital-acquired infections The presence of a glycolytic enzyme inPseudomonaswhichisknowntobe inhibitedby trehalose6-phosphate (T6P) inotherorganisms suggests that thesebacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation In the present study weexplored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P aeruginosa insupport of future target-baseddrugdiscoveryAsurveyofgenomes revealed theexistenceof2TPPgeneswitheitherchromosomal or extrachromosomal location Both TPPs were produced as recombinant proteins and character-ization of their enzymatic properties confirmed specific magnesium-dependent catalytic hydrolysis of T6P The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through b-sheet expansionof the individualmonomerswhichpossess theoverall foldofhalo-aciddehydrogenasesmdashCrossMBiberacherSPark S-Y Rajan S Korhonen P Gasser R B Kim J-S Coster M J Hofmann A Trehalose 6-phosphatephosphatases of Pseudomonas aeruginosa FASEB J 32 000ndash000 (2018) wwwfasebjorg

KEY WORDS drug discovery bull enzyme activity bull halo-acid dehydrogenase bull multidrug resistance bull proteinstructurendashfunction

Pseudomonas aeruginosa is a gram-negative multihostopportunistic bacterium that infects humans (1) livestock(2 3) plants (4) rodents insects (5) and nematodes (6) Inhealthyhumans the innate immunesystemcaneffectivelycounteract infection by P aeruginosa however patientswith compromised host defenses in particular burn vic-tims and patients who are immunocompromisedmechanically ventilated or have cystic fibrosis are par-ticularly susceptible to infection with this pathogen (1) Inthe recent past an increase in the occurrence of drug-resistant P aeruginosa strains has been observed (7) and

the lackof effective antibiotics results in apressingneed fornew therapeutics to treat infections with this pathogen

The nonreducing disaccharide trehalose belongs to agroup of so-called compatible solutes which function asosmoprotectants and thus contribute to the protection oforganisms against osmotic stress (8 9) it is also requiredfor survival at temperatures above 37degC in stress-tolerantpathogens (10) In P aeruginosa strain PA14 trehalose hasbeen identified as a virulence factor for pathogenesis inplants but not in metazoan hosts (mice flies nematodes)(11) Trehalose biosynthesis in PA14 occurs in the treYZpathway whereby oligomaltodextrins (eg glycogen) areconverted into trehalose in a 2-step reaction by maltooli-gosyl trehalose synthase (treY) and maltooligosyl treha-lose trehalohydrolase (treZ)

In addition to the treYZ pathway another 4 additionalpathways of trehalose biosynthesis have been observed inprokaryotes plants fungi and nonvertebrate animalsAmong those other pathways the so-called osmoticallyregulated trehalose synthesis (ots)AB pathway has attrac-ted particular attention as a target of interest for thera-peutic intervention in infectious diseases [reviewed inCross et al (12)] because accumulation of the metabolitetrehalose 6-phosphate (T6P) results in a lethal phenotype

ABBREVIATIONS chTPP chromosomal trehalose 6-phosphate phospha-tase ecTPP extrachromosomal trehalose 6-phosphate phosphatase GBGenBank HAD haloacid dehydrogenase MD molecular dynamics otsosmotically regulated trehalose synthesis PDB Protein Data Bank PEGpolyethylene glycol PGDB Pseudomonas Genome Database T6P treha-lose 6-phosphate TLS translationlibrationscrew TPP trehalose 6-phosphate phosphatase treY maltooligosyl trehalose synthase treZmaltooligosyl trehalose trehalohydrolase UDP uridine diphosphate1 These authors contributed equally to this work2 Correspondence Griffith University N75 Don Young Rd Nathan QLD4111 Australia E-mail ahofmanngriffitheduau

doi 101096fj201800500RThis article includes supplemental data Please visit httpwwwfasebjorgto obtain this information

0892-6638180032-0001 copy FASEB 1

ah
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there seems to be variation in the use of in house and in-house perhaps just one setting should be used throughout

in Caenorhabditis elegans and Mycobacterium tuberculosis(13 14)

The central steps of the otsAB trehalose biosyntheticpathway involve formation of T6P from uridinediphosphate-glucose and glucose-6-phosphate by the en-zymeT6P synthase (otsA EC24115 InternationalUnionof Biochemistry and Molecular Biology Calgary ABCanada) and subsequent hydrolysis of T6P by trehalose6-phosphate phosphatase (TPP otsB EC 31312 In-ternational Union of Biochemistry and Molecular Bi-ology) yielding trehalose and ortho-phosphate (15 16) Incontrast to fungi which employ cooperativemultienzymecomplexes (17) T6P synthase and TPP operate as func-tionally isolated enzymes in bacteria albeit the expressionof the corresponding genes otsA and otsB appears tightlyregulated (18 19) In Pseudomonas the otsAB pathway andits gene products have previously been investigated in thesolvent-tolerant strain Pseudomonas sp BCNU 106 (19) Inaccordance with the expected link between the otsABpathway and osmoprotection BCNU 106 displayedtoluene-induced overexpression of the genes otsA andotsB resulting in high levels of intracellular trehalose

As part of our ongoing studies of pathogen TPPs asinfectious disease targets we identified and investigatedTPP sequences from P aeruginosa as chromosomal andextrachromosomal genes With a view toward structure-based discovery of potential T6P inhibitors we investi-gated the 3-dimensional crystal structure of chromosomalPaer-TPP and characterized the enzyme activity of chro-mosomal and extrachromosomal Paer-TPP

MATERIALS AND METHODS AQ4

Mining of databases and secondarystructurendashbased alignment

Amino acid sequences of putative P aeruginosa TPPs wereidentified by database mining using the protein BLAST(BLASTp National Center for Biotechnology InformationBethesda MD USA) algorithm (20) with the sequence of Steno-trophomonasmaltophiliaTPP [CCH13862GenBank (GB)NationalCenter for Biotechnology Information] as well as a key-wordsearch for ldquotrehalose phosphataserdquo in the GB database (httpncbinlmnihgovGenbank) and Pseudomonas Genome Database(PGDB httpPseudomonascom) Secondary structure elementsfor each amino acid sequence were predicted by the softwarePSIPRED (21) installed in house A secondary structurendashbasedsequence alignment was generated automatically with the soft-ware SBAL (22) visually inspected and manually adjusted(Fig 1)

Protein expression and purification

The codon-optimized expression constructs of the chromosomal[GB NZ_JTMO01000001 (7065971417) GB WP_043516570strain AZPAE15058 (whole-genome shotgun sequence] and ex-trachromosomal [GB KC543497 (5846759225) GB WP_010792510 strain PA96 plasmid pOZ176] TPP genes of P aeru-ginosaweresynthesizedbyGenScript (PiscatawayTownshipNJUSA) and ligated into the vector p11 (The Biodesign InstituteArizona State University Tempe AZ USA) via NdeI and BamHIrestrictionsites resulting inprotein constructswithanN-terminalfusion peptide (MGSSH6SSGRENLYFQGH) Expression andpurification including proteolytic cleavage of the N-terminal

COLOR

Figure 1 Conservation of bacterial TPP sequences Structure-based amino acid sequence alignment of 18 bacterial TPP enzymesshows conservation of the characteristic HAD motifs IndashIV with key residues in bold as well as the P aeruginosandashspecific b2b3hairpin (flap-like motif) and connector helix (a3) The coloring of topological elements in line 2 is consistent with theillustration of the Paer-chTPP crystal structure shown in Fig 2 Secondary structure elements (experimentally observed for Paer-chTPP and predicted for all others) for individual sequences are mapped with green (a helix) and red (b strand) backgroundcysteine residues are highlighted in yellow GB accession numbers of TPP genes are as follows Pseudomonas aeruginosachromosomal (GB WP_043516570) and extrachromosomal (GB WP_010792510) and Arthrobacter aurescens (GB WP_011773668) Renibacterium salmoninarum (GB WP_012243900) Mycobacterium smegmatis (GB YP_890267) Thermoplasmaacidophilum (GB WP_010901616) Thermoplasma volcanium (GB WP_010917513) Acinetobacter baumannii (GB EGU03169)Escherichia coli (GB KJJ47768) Escherichia coli O157 (GB EGD67586) Shigella boydii (GB ACD06494) Shigella flexneri (GBKFZ97274) Vibrio parahaemolyticus (GB KKY41738) Shigella dysenteriae (GB WP_024250312) Shigella sonnei (GB AMG15538)Citrobacter koseri (GB WP_047464023) Salmonella enterica (GB WP_000840115) and Stenotrophomonas maltophilia (GBCCH13862) Figure prepared with PSIPRED (21) SBAL (22) and Inkscape (87)

2 Vol 32 October 2018 CROSS ET ALThe FASEB Journal x wwwfasebjorg

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(see Table 1)
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httpsinkscapeorg
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should this be italicised (consistency with web link above)
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)

fusion tag was performed according to the protocol publishedpreviously by Cross et al (23)

Selenomethionine-labeled chromosomal TPP was expressedin the auxotrophicEscherichia coli strain834(DE3)with SelenoMetmedium (Anatrace Maumee OH USA) Briefly a 2-L pro-duction culture was grown for 3 h at 37degC and induced withisopropyl b-D-1-thiogalactopyranoside (1 mM final concentra-tion) after lowering the temperature 20degC incubation at thattemperature was continued for another 20 h

The purified protein samples were dialyzed against 100 mMNaCl 1 mM MgCl2 1 mM DTT and 20 mM Tris (pH 80) andwere concentrated by ultrafiltration with an Amicon Ultracartridge (Merck Kenilworth NJ USA) with a 10-kDa cutoffAll stages of protein purificationweremonitored by SDS-PAGEconfirming the expected molecular mass of 28 kDa (Supple-mental Fig S6) The final purified nontagged proteins weresubjected to nanoliquid chromatographyndashtandem mass spec-trometry fingerprinting confirming their identity with a totalcoverage of 25 of the amino acid sequence (SupplementalTable S1AQ5 )

Determination of quaternary structure in solution

Thequaternary structureofPaer-TPPs [chromosomal trehalose 6-phosphate phosphatase (chTPP) 110 mgml extrachromo-somal trehalose 6-phosphate phosphatase (ecTPP) 104 mgml]with His-tag fusion peptides removed was assessed by size-exclusion chromatography with a buffer consisting of 100 mMNaCl and20mM4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES pH80) for isocratic elution of a Superose 12 10300GL column (GEHealthcare Life Sciences Little Chalfont St GilesBuckinghamshire United Kingdom) mounted on a DuoFlowHPLC system (Bio-Rad Laboratories Hercules CA USA) Thechromatogramswereanalyzedwith the softwareSDAR(24) andmolecularmasseswereestimated for the eluting species basedoncomparison with elution times of proteins of known molecularmass (see Supplemental Data S1)

Crystallization

The purified recombinant proteins were subjected to initialcrystallization screening (sitting drop vapor diffusion) using ourlarge in-house factorial collection with 1300 preformulatedconditions For Paer-chTPP crystals with a shape reminiscent ofarsendescloizite (25) were obtained from a range of conditionscontaining 20 polyethylene glycol (PEG PEG 3000ndash6000) andpH values between 6 and 8 within 1ndash2 wk The largest crystals(03 mm 3 1 mm) with the best diffraction properties wereobtained in hanging-drop experiments from 02 M MgCl2 20PEG 6000 01 M 2-(N-morpholino)ethane sulfonic acid (pH 6)Crystalswere cryoprotectedby flash soaking inbuffer containing25 ethylene glycol and frozen immediately in liquid nitrogenDespite extensive efforts no crystals could be obtained for Paer-ecTPP

Diffraction data collection crystal structuresolution and refinement

X-ray diffraction data from Paer-chTPP collected at the in-housediffractometer (MicroMax-007 HF R-Axis IV++ detector OxfordCryosystems 800 equipment T = 100 K Rigaku Tokyo Japan)were limited to 3 A presumably because crystals were verysensitive to the ambient humidity Diffraction data obtained atthe Pohang Accelerator Laboratory (Pohang Gyeongbuk Re-public of Korea) extended up to 19 A resolution Data sets wereindexedwithXDS(26) andscaling truncationandanalysiswere

performedwith programs from the CCP4 suite (27) Attempts tosolve the crystal structure by molecular replacement (using alibrary of 24models derived frompublished TPP structures) andheavy atom derivatization using soaking procedures were un-successful Therefore the anomalous data obtained from crystalsof selenomethionine derivatized protein were used for structuresolution Theprotocol for structure solutionby single anomalousdiffraction as implemented in Auto-Rickshaw (28) (beamtimemode EMBL-EBI European Molecular Biology LaboratoryndashEuropean Bioinformatics Institute Hinxton United Kingdom)was used to initiate substructure determination and initial phasecalculation for data set TPP022 at a resolution of 37 A AQ6with theSHELXCDE set of programs (29) Sixteen heavy-atom siteswere found and the correct hand for the substructure was de-termined using the programs ABS (30) and SHELXE The occu-pancy of all substructure atoms was refined using the programBP3 (31) The 2-fold noncrystallographic-symmetry operatorwas found with the RESOLVE program (32) Density modifica-tion phase extension and noncrystallographic symmetryndashaveragingwere performedwith theprogramDMfrom theCCP4suite resulting in the localizationof 22heavy-atomsitesApartiala-helical model was produced with ARPwARP (33) and ex-panded by iterative rounds of manual model building andcomputational refinement Once the backbone of 470 of 508 res-idues (93) had been traced and a reasonable number of aminoacid side chains had been built the model was used to solve thestructure of data set TPP023 by molecular replacement Furtheriterative cycles ofmanualmodel adjustments and computationalrefinement enabled buildingof amodel for all but the last residue(Glu252) in both molecules of the asymmetric unit Analysis ofpossible rigid-body displacements in themodel with anisotropicB-factors with the TLSMD server (34) allowed identification ofthe top 3 groups of translationlibrationscrew (TLS) motionsper monomer which were included in the computational re-finement All manual model building was performed with Coot(35) and O (36) and computational refinement of atomic posi-tions atomic displacement factors and TLS groups was donewith Phenix (37) For data collection phasing and refinementstatistics (see Table 2 AQ7) The dimer interface was analyzed usingthe PISA web service (EMBL-EBI) (38) Structure factors andatomic coordinates of the refined structure of Paer-chTPP (dataset TPP023) have been deposited with the Protein Data Base(PDB accession number 6cj0)

Modeling of substrate-bound Paer-chTPP

The substrate-bound structure of Paer-chTPP was modeled bymanually docking T6P into active site of the protein using thecrystal structure described in this study Force field parametersfor T6P were generated with the PRODRG2 server and a mo-lecular dynamics simulation of the solvated complex was per-formed with Gromacs 465 the Gromos 43a1 force field and aTIP3P water model (39) To ensure charge-neutrality and anelectrolyte concentration of 100 mM sodium and chloride ionswere added to the octahedral cell by replacing solventmoleculesAfter an energy-minimization step a position-restrained dy-namic simulationof 20pswasperformed tograduallyequilibratethe solvated complex at 300 K and 1 bar Periodic boundaryconditions were applied in all 3 dimensions Long-range inter-actionsweremodeledusing theparticlemeshEwaldmethod (40)and a grid spacing of 12 A the cutoff for computation of short-range electrostatic interactionswas10 A andwas 14 A forvanderWaals interactions The temperature was controlled with theV-rescale thermostat (41) and the pressure was controlled withthe Parrinello-Rahman barostat (42) bonds were constrainedwith the LINCS algorithm (43) The final molecular dynamics(MD) simulation was performed for 30 ns with a time step of0002 ps The simulationwas performed on a custom-built server

TPP OF PSEUDOMONAS AERUGINOSA 3

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with a Xeon E5-1650 6 Core (35 GHz Intel Santa Clara CAUSA) and 32 GB random access memory (RAM) Analyses wereperformed with Gromacs tools and automated plots were gen-erated with Grace (44)

Chemicals

T6P was synthesized in house as published previously byCross et al (23) and was purchased from Santa Cruz Bio-technology (Dallas TX USA) Trehalose 6-sulfate was syn-thesized in house following the published procedure of Farelliet al (45) Flavomycin ADP and phosphosaccharides wereobtained from MilliporeSigma (Billerica MA USA) and uri-dine diphosphate (UDP) and UDP-glucose were purchasedfrom Abcam (Cambridge United Kingdom) All otherchemicals were resourced from MilliporeSigma unless oth-erwise stated

Enzyme kinetics assays

Phosphatase activity of purified recombinant Paer-TPPs wasassessed at a final enzyme concentration of 10 mM in a buffersolution containing 100 mM NaCl and 20 mM Tris (pH 75) aswell asvaryingconcentrationsofT6P (0ndash40mM)Reactionswereset up in a total volume of 180 ml in 96-well plates (CorningCorning NY USA MilliporeSigma) at room temperature star-ted by the addition of the enzyme and stopped at 30-s to 1-minintervals by transferring 25ndash50 ml of reaction mix into 200 mlBiomol Green reagent (Enzo Life Sciences Farmingdale NYUSA) After incubation for 15min the absorbance at 620 nmwasmeasured using a plate reader (BioTek Instruments WinooskiVT USA) All reactions were set up in triplicate and controlexperiments in the absence of enzyme were used to correct forbackground absorbance The corrected data were converted tomolar concentration of phosphate using a calibration functionthat was determined for every new batch of Biomol Green Afterassessment of the raw data with SDAR (24) we concluded thatmodeling of burst-like kinetics (if present) was not feasible be-cause of the lowmagnitude of the observed spectroscopic signalDatawere thus analyzed by extracting initial rateswith linear fitsof the raw data using R software (R Foundation for StatisticalComputing Wien Austria) (46)

Enzyme end-point assays

Phosphatase end-point assays were used to assess possiblesubstrates of Paer-TPPs as well as inhibitors of T6Pase ac-tivity Enzyme activity was assessed at fixed substrate andenzyme concentrations (500 and 10 mM respectively) in 50-ml reaction mixtures in assay buffer [100 mM NaCl 20 mMTris (pH 75)] Potential inhibitors were added to the enzymeat a final concentration of 1 mM and the mixtures were in-cubated for 5 min before reactions were initiated by the ad-dition of T6P

Reactionswereallowed toproceed for6minbeforequenchingwith 100 ml of BIOMOL Green reagent Absorbance at 620 nmwas determined using a plate reader (BioTek Instruments) afteran incubation period of 15 min for color development All reac-tions were set up in triplicate in 96-well plates and control ex-periments in the absence of enzyme were used to correct forbackground absorbance

RESULTS

Genomic identification of Paer-TPP andsequence comparison with bacterial TPPs

A survey of genomic databases available through GB andthe PGDB showed that P aeruginosa possesses 2 TPPproteins 1 with chromosomal and 1 with extrachromo-somal location (Table 1) Notably the occurrence of bothproteins is mutually exclusive all P aeruginosa strains forwhich informationwas available to date possess either thechromosomal (Paer-chTPP) or the plasmid-encoded (Paer-ecTPP) T6P

Comparisonof thePaer-TPP sequenceswith aminoacidsequences from other bacterial TPPs highlighted the con-servation of the canonical haloacid dehydrogenase (HAD)motifs (IndashIV) and revealed the presence of P aeruginosandashspecific insertions between motifs I and IV as well as thefirst linker region between the core and the cap domain(Fig 1)

TABLE 1 TPP genes in P aeruginosa identified in genomic databases

Chromosomal TPP Extrachromosomal TPP

Strain Accession noIdentity querysequence () Strain Accession no

Identity querysequence ()

AZPAE15058 GB NZ_JTMO01000001 100 PA96 plasmid pOZ176 GB KC5434971 10029785cz GB KY8605721 99 FFUP_PS_37 plasmid pJB37 GB KY4948641 99PA58 GB CP0217751 99 PA121617 plasmid pBM413 GB CP0162151 99

PGDB NZ_CP016215 99E6130952 GB CP0206031 99H47921 GB CP0088611 99

PGDB NZ_CP008861 100VR-14397 GB LK0545031 99NCGM1984 GB AP0146461 99

PGDB NZ_AP014646 100NCGM1900 GB AP0146221 99

PGDB NZ_AP014622 10037308 GB GQ1618471 99PACS171b GB EU5957501 99PA1207 GB CP0220011 9939016 PGDB NZ_CM001020 100


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httpplasma-gateweizmannacilGrace
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httpswwwr-projectorg
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also

Quaternary solution structure of Paer-TPPs

The quaternary solution structure of both Paer-TPPs wasassessed by size-exclusion chromatographyAlthough therecombinant chromosomal TPP eluted exclusively as adimeric species amonomerndashdimermixturewith a ratio of11 was observed for the extrachromosomal TPP

Crystal structure of chromosomal Paer-TPP

The crystal structure of Paer-chTPP (diffraction data andrefinement statistics are summarized in Table 2) showsthat the protein adopts the general fold known from othermembers of the HAD superfamily (InterPro IPR023214EMBL-EBI) of enzymes The overall foldwhich comprises

TABLE 2 Crystallographic data collection and refinement statistics for Paer-chTPP

Se-Met

TPP022 TPP023g

Parameter Total Total Molecule 1 Molecule 2

DatAQ15 a collectionX-ray source PAL MX-7A PAL MX-7ADetector ADSC Q270r ADSC Q270rWavelength (A) 09793 09793Space group P21 P21

Cell dimensionsa b c (A) 748 669 754 749 670 756a b g(deg) 90 1197 90 90 1197 90Resolution (A)a 25ndash23 (242ndash230) 25ndash19 (100ndash190)Unique reflectionsa (n) 28778 (4126) 51243 (5069)Rsym

ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim

a 0025 (0086) 0042 (0219)CC(102)a 0999 (0981) 0997 (0898)Mean IsIa 209 (78) 106 (36)Completenessa 0996 (0989) 0997 (0998)Redundancya 75 (74) 75 (75)Wilson B factor (A2) 378 257

PhasingHeavy atom sites (n) 22Correlation coefficient (all weak)c 298 129Mean figure of merit 0701

RefinementResolution (A) 25ndash19 (197ndash190)Reflections (n) 51236 (5069)Working seta 48676 (4784)Test seta 2560 (285)

Atoms (n)Protein 3954 1977 1977Ligandion 2 Mg2+ 2 CO3

22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22

Water 215Average B-factorsProtein (A2) 419 419 419Ligandion (A2) 274 279 269Water (A2) 324

Root mean square deviationsBond lengths (A) 0007Bond angles (deg) 0979B factor for bonded atoms 586 590 590

MolProbity analysisd

Ramachandran outliers () 04Ramachandran allowed () 28Ramachandran favored () 968Rotamer outliers () 31Cb outliers 0Clash score 421R factorae 0174 (0226)Rfree

af 0218 (0301)

aValues in parentheses are for highest-resolution shell bRsym = S |I ndash I|S Ι where I is the observed intensity and I is the averageintensity obtained from multiple observations of symmetry-related reflections after rejections cDefined in Schneider and Sheldrick (85) dResultsfrom the MolProbity analysis as implemented in Phenix (37) eR-factor = S | |Fo|ndash|Fc| |S |Fo| where Fo and Fc are the observed and calculatedstructure factors respectively fRfree is defined in (86) gPDB accession number 6cj0


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the core domain with a Rossmann fold and an ab capdomain allows for subtle variations when comparingdifferent members of this protein family AccordinglyHAD proteins can be classified into 3 subtypes based onthe structure and insertion location of the cap domainTwo sites for cap insertion exist 1 in the middle of theb-hairpin formedbyb-strands 2 and3 (C1) and another inthe linker after b-strand 3 of the core domain (C2) thosemembers of the protein family having only small inser-tions at either site are designated as C0 C1 caps may beeither a-helical or assume the unique ab fold associatedwith P-type ATPases whereas C2 caps have a distinctb-sheet core surrounded by ab elements (47ndash49)

The crystal structure of Paer-chTPP confirms the pres-ence of all structural elements defining the HAD foldthe sequence of secondary structure elements is N-bbbababba(abbabb)ababa-C indicating that the pro-tein belongs to the HAD C2 subfamily (brackets indicatethe cap domain the underlined a-helix is a contiguouselement linking the core and cap domain) The coredomain is constituted by a 3-layer aba sandwich withthe central 6-stranded b sheet comprising b12(uarr)-b11(uarr)-b1(uarr)-b4(uarr)-b5(uarr)-b6(darr) The cap domain is situatedjuxtaposed and connected to the core domain by the longbent-helixa3 and a coiled linker segment betweenb10 anda5 Like in otherHADC2 familymembers the capdomainof Paer-chTPP possesses abbabb topology with a4-stranded antiparallel b sheet (b7-b8-b10-b9) flanked by

2 a helices (a3 a4) on the side facing away from the coredomain

Paer-TPPs possess 2 obvious differences and to ourknowledge previously unobserved structural elementscomparedwithotherTPPstructures first thepresenceof ab hairpin comprising b-strands b2 and b3 and second along bent helix (a3) that connects the HAD core with thecap domain (Fig 1)

The crystal structure of Paer-chTPP reveals that the bhairpin extends from the core domain (see Fig 2) andnarrows theaccess to the substrate-bindingpocketCrystalstructures of other bacterial and nematode TPPs (Thermo-plasma acidophilumTPP PDB 1u02BrugiamalayiTPP PDB4ofz 5e0o) confirm the absence of thatb hairpin howevera conserved structural element in that place is common toother HAD family proteins and is known as the ldquoflapmotifrdquo (47)Movement of the flapmotif aswell as a helicalturn situated immediately upstream controls access to theactive site particularly in HADs with immobile cap do-mains (47) An intriguingly novel structural elementrevealed by the crystal structure of Paer-chTPP is the firstconnector between the HAD core and the cap domainAlthough the segments connecting the cap and core do-mains in TPPs are typically short extendedunstructuredloops theN-terminal connector is replaced here by a longbent a-helix (a3) with Trp111 (observed in 2 alternateconformations) at the bending point This connector foldconstitutes a rather rigid attachment of the cap to the core

COLOR

Figure 2 The crystal structureof Paer-chTPP A) Stereo figureof a cartoon representation ofthe dimer in the X-ray crystalstructure The HAD core do-main with the characteristicRossmann fold is blue and theHAD cap domain is teal Theb2b3 hairpin (flap-like motif)is highlighted in yellow and thebent helix (a3) connectingcore and cap domain is shownin turquoise The location ofHAD motifs IndashIV is shown inorange (I II) and green (IIIIV) and magnesium ions arerendered as magenta spheresB) Stereo figure of the 2FondashFc(contoured at 1 s) electrondensity in the active site show-ing the octahedral coordinationof the protein-bound magne-sium ion (magenta sphere)Coordination is provided by theside-chain carboxylate groups ofAsp11 Asp14 and Asp220 thebackbone carbonyl of Asp13 2water molecules (red spheres)and a bound ligand modeled ascarbonate Figure prepared withPyMOL (88) and Inkscape (87)


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-helical

domain because of the inherent directional stability of thehelical fold

Notably the crystal structure of Paer-chTPP reveals ahomodimer inwhich 2monomersmake contact through apredominantly hydrophobic interface around b12 andb129 In the dimer the centralb sheet of the core domain isthus extended to a 12-stranded b sheet and the dimerinterface is further expanded by the formation of a 4-helixbundle comprising a6 a7 a69 and a79 (Fig 2)

T6P-bound structure of chromosomal Paer-TPP

To gain insights into the molecular interactions betweenthe substrate and the enzyme we docked T6P into theactive site of Paer-chTPP and subjected the hydratedcomplex to a molecular-dynamic simulation of 30000 psduration The simulation revealed that a stable-bindingpose of the substrate in the active site can be obtained (seeSupplemental Fig S1 and Table 3)

TheMDsimulationhighlighted substantial flexibilityofthe b2b3 hairpin with respect to the core domain Thatflexibility is illustrated by the highly variable distance be-tween the hairpin tip and the end-cap residue of helix a1(see Supplemental Fig S1AQ8 ) A comparison of the confor-mationof theb2b3hairpin in the crystal structureand theMD simulation at 30000 ps (Fig 3A B) shows that thehairpin closes over the active site when the substrate isbound Notably the distance between the centers ofgravity of the cap and core domains remained ratherconstant (between 21 and 23 A see Supplemental Fig S1)throughout the duration of the simulation thereby in-dicating that the openingclosingmovement between thecap and core domains as observed in other TPPs is likelyabsent in the P aeruginosa enzymes (Fig 3A B)

In the active site the substrate adopts a curved-conformation wound around a ldquolockrdquo between the coreand the cap domain formed by the side chains of Asp14and Asn182 (Fig 3B and Supplemental Fig S1) The T6Pgroup coordinated the protein-boundmagnesium ion andmaintained a stable contact with the side chain hydroxylgroup of Ser221 (Supplemental Fig S1) Although the 3-hydroxy group of T6P was engaged in a hydrogen bondwith the backbone carbonyl of Asn185 from early onduring the simulation the hydroxy groups of the second

glucose moiety formed interactions with the protein onlyat times beyond 25000 ps specifically hydrogen bondswere observed between the 29-hydroxy group and thebackbone amine of Asp14 the 49-hydroxy group and theside chain hydroxy group of Thr19 and the 69-hydroxygroup and the side chain carbonyl of Asn185 (Supple-mental Fig S1)

Compared with the crystal structure in which themagnesium ion was coordinated by the side-chain car-boxylates of Asp11 and Asp220 as well as 2 water mole-cules anda carbonate ion (Fig 2) themetal ion relocated inthe simulation of the T6P-bound protein and remainedstably coordinated by the T6P group the side chain car-boxylates ofAsp220 and 2watermolecules The side chainofAsp11 appeared not involved in direct interactionswiththe substrate

Enzyme activity of Paer-TPPs

Phosphatase activity of both recombinant Paer-TPPs wasassessed with T6P and a panel of phosphocarbohydratesThe panel consisted of the nucleoside phosphates ADPUDP and UDP-glucose as well as glucose 6-phosphatesucrose 6-phosphate galactose 6-phosphate fructose 16-bisphosphate glucose 16-bisphosphate and T6P Amongthe compounds tested notable phosphatase activity wasobserved only for T6P (Fig 4) Enzymatic activity of bothPaer-TPPs relies on the presence of magnesium ions withanoptimumpHof65ndash7 notably aplot of enzymeactivityagainst pH yields bell-shaped curves for both enzymes(Supplemental Fig S2) in agreement with the expectedinvolvement of 2 catalytically active aspartate residues inthe enzymatic mechanism of HAD enzymes The experi-mentally determined pKa values for the catalytic residueswere615and71 forPaer-chTPPand645and725 forPaer-ecTPP (Supplemental Fig S2) Those values are muchhigher than the pKa value for the side chain of free asparticacid However such an increase in pKa values for acidiccatalytic residues is frequently observed and is a conse-quence of the particular environment within a foldedprotein (50 51)

The T6Pase kinetics of both proteins revealedMichaelis-Menten behavior (Fig 5) albeit with low effi-ciency and large KM values (Table 4) Notably despite

TABLE 3 Ligandndashprotein interactions in simulated Paer-chTPPT6P complex

Ligand Protein Distance (A)a

PO4-OAX Mg2+ 18PO4-OAY Mg2+ 18PO4-OAY (SOL-4233 SOL-3507) rarr D11-OD1 26 29 rarr (28 27)PO4-OAX D220-OD2 33PO4-OAY D220-OD1 30PO4-OAX S221-OG 30PO4-OAY S221-OG 253-OH N185-CO 3329-OH D14-NH 3349-OH T19-OG 3269-OH N185-OD1 36

aAt simulation time t = 30000 ps


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Paer-chTPP forming a dimeric species no cooperativitywas observed in the plot of the initial rate vs the substrateconcentrationAlthoughother bacterial andnematodeTPPshave previously been shown to possess burst-like kinetics(52) a clear conclusion to that effect cannot be made for the2 Paer-TPPs because of the low turnaround of substratewhich resulted in a spectroscopic signal of lowmagnitude

To assess susceptibility of Paer-TPPs to common phos-phatase inhibitors and probe molecules a panel of 8commercially available compounds and the in-house

synthesized sulfate analog of T6P trehalose 6-sulfatewere tested at a concentration of 1 mM in competitiveinhibition assays against T6P as the substrate (Fig 6) In-hibitors that act by restricting access to the catalytic metalion showed no substantial effects on the T6Pase activity ofthe 2 Paer-TPPs In contrast the presence of the divalentmetal chelatorEDTAor the sulfateanalogofT6P trehalose6-sulfate led to significant reduction in enzymatic activityby 70ndash80 Notably the antibiotic Flavomycin increasedphosphatase activity

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Figure 3 Docking of T6P into the crystal structure of Paer-chTPP T6P was docked into the crystal structure of Paer-chTPP andthe hydrated complex was subjected to MD simulation Comparison of the surface representations of the crystal structure (A) anda snapshot from the MD simulation at t = 30000 ps (B) shows that the relative orientation of core (blue) and cap (gray) domainsdo not change The notable conformational changes upon substrate binding include a closing of the active-site cleft by the b2b3hairpin (cyan) and the formation of a ldquolockrdquo by the side chains of Asp14 and Asn182 C) Cut-away view of the active-site cleft withT6P rendered as a stick model and magnesium shown as a magenta sphere direct interactions between substrate and protein areindicated (see also Table 3) Figure prepared with PyMOL (88) and Inkscape (87)

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Figure 4 The phosphatase activity of recombinant Paer-TPPs is specific for T6P Comparison of corrected absorbance data (l =620 nm) obtained in endpoint assays of recombinant Paer-chTPP (blue) and Paer-ecTPP (red) with nucleoside phosphates (left)and other carbohydrate phosphates (right) Data shown represent the means of 3 technical repeats error bars indicate the SEFigure were generated with R software (46) and Inkscape (87) Left) The nucleoside phosphates ADP UDP and UDP-glucosewere tested at 200 mM concentration in endpoint assays containing 25 mM enzyme For comparison activity observed with calfalkaline intestine phosphatase (CIP M0290 New England BioLabs Ipswich MA USA) is shown in green Right) Thecarbohydrates glucose 6-phosphate sucrose 6-phosphate galactose 6-phosphate glucose 16-bisphosphate and fructose 16-bisphosphate were tested at 500 mM concentration in endpoint assays containing 10 mM enzyme


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DISCUSSION

P aeruginosa possesses a chromosomal andan extrachromosomal TPP

A survey of genomic databases revealed that P aeruginosastrains possess genes coding for 2 different TPPs Theamino acid sequences of the 2 TPP gene products share93 identity Although 1 of the 2 TPPs originated from agene that hadbeenmapped on the chromosome the otherTPP gene was located on a plasmid Interestingly in all Paeruginosa strains with available genome information theexistence of either chromosomal or extrachromosomalTPP is exclusive

The dynamic pathogenicity and impressive adaptabil-ity of the Pseudomonas species has been attributed to theirmosaic genomic structure which comprises a conservedldquocorerdquo genome and a variable ldquoaccessoryrdquo genome (53)The accessory genome varies widely among strains and isassociated with the bacteriumrsquos genetic plasticity andsurvival in specific environmental niches (53 54) Becausethose advantageous genes are believed to be acquiredthrough horizontal gene transfer they have been dubbedldquogenomic islandsrdquo and may take the form of plasmidsconjugative transposons nonreplicative elements orcryptic prophages (55) The transfer of plasmids from Ecoli toP aeruginosahaspreviously beendemonstrated (56)and the presence of an otsB gene in different genomic lo-cales in P aeruginosa suggests that it may have been ac-quired by different strains when required to confer afitness advantage Many Pseudomonas species express thetreP phosphotransferase subunit and the treA trehalasewhich allow the importation and use of environmentaltrehalose as an energy source (57 58) respectively and

treA has been annotated in the P aeruginosa genome(UniProt Q9I165) Building on this existing component ofthe trehalose catabolism the ability to synthesize this di-saccharide could provide additional benefits such as os-moregulation and abiotic stress tolerance to P aeruginosaas in other bacteria (10)

Theenclosedarea in thedimer interfaceofPaer-chTPP is1350 A2 and thus11of the total solvent-accessible areaof 1 monomer suggesting that the dimer observed in thecrystal structure may be of physiologic significance (59)This hypothesis is further supported by data from size-exclusion chromatography which revealed a 66 kDa (di-mer) species as the predominant form of the protein insolution at pH 8 (Supplemental Fig S3) Although the di-mer formation with the characteristic motif of b-sheet ex-pansion has not been observed so far with TPPs thatbelong to the HAD C2 subfamily (see above) there isprecedence for this type of homo-oligomerization withinthe larger HAD family of proteins In particular somesubtype C1 HAD members exhibit similar dimers in-cluding the cytosolic 59-(39)-deoxyribonucleotidase (PDB2jar) mitochondrial 59-(39)-deoxyribonucleotidase (PDB2jaw) and soluble epoxide hydrolase 2 (PDB 5ai0)

Although the sequences of both TPPs differ by only 18aa substitutions (Fig 7) those variations result in a changein the quaternary structure in solution Although Paer-chTPP exists exclusively as a dimeric species in solutionPaer-ecTPP forms a roughly equimolar mixture of mono-mers and dimers (Supplemental Fig S3) This hetero-dispersive behavior in solution may be one reason for thefailure to obtain crystals of Paer-ecTPP Analysis of thedimer interface (mainly around the C-terminal b strandand a helix) shows that none of the amino acid substitu-tions in the plasmid-encoded TPP as compared with thechromosomal TPP are located in that area The only var-iable amino acids in the dimer interface comprise Ile247(Val in ecTPP) andGly229 (Asp in ecTPP) neither ofwhichis engaged in particularly significant interactions In-triguingly 3 of the variation hotspots in the P aeruginosaTPPs are involved in the crystal contacts of thedimer in thecrystal structure of Paer-chTPP Ser53 (Asn in ecTPP)Lys108 (Glu in ecTPP) and Arg126 (Gln in ecTPP) areinvolved in lattice contacts with Thr30 Trp111 andGlu247 respectively Of those 3 contacts the 1 most likelyto be perturbed by the amino acid variation betweenchromosomal and plasmid-encoded TPP is Lys108Trp111 Formation of a hydrogen bond between the sidechain amino group of Lys108 and the aromatic system ofTrp111 is not possible if the lysine is replaced with a glu-tamate residue

COLOR

Figure 5 Steady-state Paer-TPP enzyme kinetics follow Michae-lis-Menten behavior A plot of initial rates of phosphataseactivity of Paer-chTPP (blue enzyme concentration 5 mM) andPaer-ecTPP (red enzyme concentration 10 mM) at varying T6Pconcentrations reveals Michaelis-Menten behavior The solidlines represent the fits of the Michaelis-Menten equation usingR software (46) Numerical fitting results are listed in Table 4

TABLE 4 Michaelis-Menten parameters for T6Pase activity ofPaer-TPPs

Parameter Paer-chTPP Paer-ecTPP

Km 32 mM 42 mMkcat 006s 006sh = kcatKm 19sM 14sM

KM and kcat were obtained from fitting the Michaelis-Mentenequation to the data shown in Fig 5


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Enzyme activity of P aeruginosa TPPs highsubstrate specificity but moderate efficiency

From a panel of 8 metabolically relevant phosphocarbo-hydrates phosphatase activity was observed only withT6P (Fig 4) confirming the anticipated function of the 2 Paeruginosa enzymes as TPPs and the high substrate speci-ficity of those enzymes Compared with the enzymatic

activities of all other known TPPs Paer-TPPs possess thelowest efficiency so far with h values of 10ndash20sM(Table 4) which is 1 order of magnitude less than the ef-ficiency observed for TPP from Stenotrophomonas malto-philia (52)

The low enzyme efficiency arises for 2 reasons a ratherhighKMand an extremely low catalytic rate constant (kcat)The unusually low turnover rate is a consequence of thespecific structural fabric of the enzyme Particularly thepresence of a flap-like element in the form of the b2b3-hairpin provides spatial restrictions at the entry to thesubstrate binding site Additionally the rigid attachmentof the cap to the core domain by means of helix a3 mayprevent conformational movements between the 2 do-mains (ie ldquoopeningrdquo and ldquoclosingrdquo) and thus force a veryspecific entryexit trajectory for reactants and productsThis hypothesis is supported by the absence of domainmovements in the MD simulation of substrate-bound Paeruginosa TPP

The steady-state kinetic parameters obtained for the Paeruginosa TPPs are very different from the kinetically su-preme enzymes typically portrayed in textbooks withtheoretical considerations suggesting an upper limit forkcat of 10

6ndash107s (60) (the upper limit for rates of diffusion-controlled reactions is 108ndash109Ms) Notably it has pre-viously been suggested that low enzyme efficiencies mayreflect the searchof a rare enzymeconformation thatyieldsthe catalytically competent substrate-bound state (61 62)Furthermore in contrast to the broadly held view thatenzymes have evolved to maximize their efficiency otherhypotheses have been presented including the idea thatevolution forges the KM value of an enzyme to suit thephysiologic substrate concentration (63) In that context asurvey of some 1000 enzymes demonstrated that moder-ate enzyme efficiencies are rather common and not anexception the kinetic parameters of the ldquoaveragerdquo enzyme

COLOR

Figure 6 The T6Pase activity of recombinant Paer-TPPs isinhibited by EDTA and trehalose 6-sulfate Comparison of therelative phosphatase activity of 10 mM of recombinant Paer-chTPP (blue) and Paer-ecTPP (red) with 500 mM T6P as thesubstrate in the presence of 1 mM of generic phosphataseinhibitors as well as Flavomycin and trehalose 6-sulfate Datashown were obtained from endpoint assays and represent themeans of 3 technical repeats error bars indicate the SE Figuregenerated with R software (46) and Inkscape (87)

COLOR

Figure 7 Structural mapping of amino acid sequence differences between Paer-TPPs Amino acid differences betweenchromosomal and extrachromosomal TPPs are mapped onto the structure of the Paer-chTPP monomer shown as cartoonrepresentation in stereo The 2 sequences are 93 identical and differ in 18 residues of 251 (7)AQ14 Figure created with PyMOL(88) and Inkscape (87)


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adopt much lower values than the upper limit and globalanalysis yieldedanaverageof kcat of10sandanaverageefficiency of 105Ms (64) Such large deviations fromthe maximum possible performance indicate that thoseenzymes are under much weaker evolutionary-selectionpressure than enzymes with high-performance parame-ters Fulfilling roles in the secondary metabolism theweaker selectionpressure facedbyTPPs fromP aeruginosaand other bacterial organisms may be due to their limitedcontributions to the fitness of the entire organism or in-deed because their activity is only required under specificconditions andor for limitedperiods iewith lowoverallturnaround (64)

Inhibition of P aeruginosa TPPenzyme activity

The inhibition profile of a panel of generic phosphataseinhibitors observed with P aeruginosa TPPs is strikinglysimilar to that observed with S maltophilia (52) AlthoughPaer-chTPP is largely unaffected generic phosphatase in-hibitors exert only minor effects on the activity of Paer-ecTPP (Fig 6) In the presence of themetal chelator EDTAhowever the enzymatic activity of both P aeruginosa en-zymes is strongly suppressedbecauseof theremovalof thecatalytic metal ion

Because synthetic carbohydrate chemistry is challeng-ing the development of substratemimics that can be usedas probe molecules and for structure-based inhibitor de-sign is slow but some progress has been made (65) Tre-halose 6-sulfate has previously been shown to marginallylimit the enzymatic activity of TPP from the nematodeBrugia malayi (45) In contrast to other nematode and bac-terial TPPs fromour in-house panel (52) thePaer-TPPs aresusceptible to trehalose 6-sulfate with the remaining ac-tivities between 20 and 30 (Fig 6)

The antibiotic complex consisting of moenomycins Aand C known as Flavomycin has previously been testedas effectors ofmycobacterial TPPs (66)We thus includedFlavomycin in the panel of potential inhibitors in the cur-rent study Similar to the observations of the TPP fromMycobacterium tuberculosis which displayed increasedT6Pase activity in the presence of up to 60mMFlavomycin(66) we found that the presence of 1 mM Flavomycin in-creased the enzymatic phosphatase activity of both Paeruginosa TPPs2- to 5-fold (Fig 6)

CONCLUSIONS

A BLAST search with the P aeruginosa b2b3-hairpin se-quence identified ldquotrehalose phosphataserdquo and ldquohypo-theticalrdquo enzymes with a very high degrees of identityfrom various Pseudomonas and Burkholderia species Asan example the amino acid sequence alignment of Paer-chTPP with trehalose phosphatase of Burkholderiaubonensis (UniProt WP_0717619711) both share the b2b3-hairpin sequence with 100 identity revealing 79identity overall (Supplemental Fig S4) Like PseudomonasBurkholderia spp are opportunistic bacteria and includethe human pathogens Burkholderia cepacia (causing

pulmonary infections) and Burkholderia pseudomallei(causing melioidosis) Intriguingly both genera make useof genomic islands for pathogenicity (67) are susceptibleto the same phage-transfer mechanisms (68) and are eachcapable of influencinggene expression in theother (69 70)MoreoverBurkholderia spppossess a full suite of trehalosemetabolismgenes including trehalasewhichsignificantlyaffects virulence and biofilm formationwhen it is knockeddown (71) This suggests an important role for trehalosemetabolism in Burkholderia [as discussed by Schwarz andVanDijck (72)] and thus potentially in Pseudomonas giventhe similarity between the 2 enzymes and the fact that thespecies occupy similar environmental niches

The toxicity of intracellularly accumulating T6P hasbeen attributed directly to its role in regulating energymetabolism and consequent ability to inhibit hexokinase(13 73ndash77) Additionally high intracellular concentrationsof this metabolite induce dysregulation of multiple genesin some cases through inhibition of SNF1-related proteinkinase 1 (14 78ndash80) The P aeruginosa enzyme originallybelieved to be hexokinase (81) was found to be inactivetoward fructose and mannose and has since been reclas-sified as glucokinase (82) Nevertheless it performs thephosphorylation of glucose to glucose 6-phosphate asyeast hexokinase does furthermore it possesses the keyresidues used by yeast hexokinase (83 84) (SupplementalFig S5) Thus although the effects of T6P accumulation inP aeruginosa are unknown the broad functionality of T6Pin signaling and regulation its widespread toxicity andthe presence of a glycolytic enzyme inPseudomonas similarto that which this metabolite is already known to inhibitall suggest that the bacterium may be vulnerable to thedetrimental effects of T6P accumulation (85ndash88) AQ9

ACKNOWLEDGMENTS AQ10

Research in the investigatorsrsquo laboratories was funded bythe Australian Research Council (to AH and RBG) theRebecca L Cooper Medical Research Foundation (to AH)and the Chonnam National University (2015-0597 to J-SK)The Equity Trustees PhD Scholarship and the AustralianGovernment Research Training Program Scholarship (to MC)as well as support by DAA AQ11D RISE and PROMOS scholarships (toSB) are gratefully acknowledged Mass spectrometric analysiswas undertaken at the Australian Proteome Analysis Facility andthe infrastructure was provided by the Australian Governmentthrough the National Collaborative Research InfrastructureStrategy The authors declare no conflicts of interest

AUTHOR CONTRIBUTIONS

M Cross R B Gasser J-S Kim M J Coster and AHofmann designed the research M Cross S BiberacherS-Y Park and S Rajan performed the experiments MCross S Biberacher S-Y Park P Korhonen R B Gasserand A Hofmann analyzed the data and M Cross and AHofmann wrote the paper with critical input from allauthors


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the German Academic Exchange Service through
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Please insert at appropriate location1313NOTE ADDED IN PROOF13Coordinates and structure factors of two related crystal structures have been deposited with the PDB (accession codes 6d3w 6d3v)

REFERENCES

1 Sadikot R T Blackwell T S Christman J W and Prince A S(2005) Pathogenndashhost interactions in Pseudomonas aeruginosapneumonia Am J Respir Crit Care Med 171 1209ndash1223

2 Leitner G and Krifucks O (2007) Pseudomonas aeruginosa mastitisoutbreaks in sheep and goat flocks antibody production andvaccination inamousemodelVet Immunol Immunopathol119 198ndash203

3 Bhatt V D Ahir V B Koringa P G Jakhesara S J Rank D NNauriyal D S Kunjadia A P and Joshi C G (2012) Milkmicrobiome signatures of subclinical mastitis-affected cattle analysedby shotgun sequencing J Appl Microbiol 112 639ndash650

4 Rahme L G Stevens E J Wolfort S F Shao J Tompkins R Gand Ausubel F M (1995) Common virulence factors for bacterialpathogenicity in plants and animals Science 268 1899ndash1902

5 Jander G Rahme L G and Ausubel F M (2000) Positivecorrelation between virulence of Pseudomonas aeruginosa mutants inmice and insects J Bacteriol 182 3843ndash3845

6 Tan M W Rahme L G Sternberg J A Tompkins R G andAusubel F M (1999) Pseudomonas aeruginosa killing ofCaenorhabditis elegans used to identify P aeruginosa virulence factorsProc Natl Acad Sci USA 96 2408ndash2413

7 Moore N M and Flaws M L (2011) Antimicrobial resistancemechanisms in Pseudomonas aeruginosa Clin Lab Sci 24 47ndash51

8 Roder A Hoffmann E Hagemann M and Berg G (2005)Synthesis of the compatible solutes glucosylglycerol and trehalose bysalt-stressed cells of Stenotrophomonas strains FEMSMicrobiol Lett 243219ndash226

9 Arguelles J C (2000) Physiological roles of trehalose in bacteria andyeasts a comparative analysis Arch Microbiol 174 217ndash224

10 Zeidler S Hubloher J Schabacker K Lamosa P Santos H andMuller V (2017) Trehalose a temperature- and salt-induced solutewith implications in pathobiology of Acinetobacter baumannii En-viron Microbiol 19 5088ndash5099

11 Djonovic S Urbach J M Drenkard E Bush J Feinbaum RAusubel J L Traficante D Risech M Kocks C Fischbach M APriebe G P and Ausubel F M (2013) Trehalose biosynthesispromotesPseudomonas aeruginosapathogenicity inplantsPLoSPathog9 e1003217

12 Cross M Rajan S Biberacher S Park S-Y Coster M J DługoszE Kim J-S Gasser R B and Hofmann A (2017) Trehalose-6-phosphate phosphatase as a broad-spectrum therapeutic targetagainst eukaryotic and prokaryotic pathogens Emerg Top Life Sci 1675ndash683

13 Kormish J D and McGhee J D (2005) The C elegans lethal gut-obstructed gob-1 gene is trehalose-6-phosphate phosphatase DevBiol 287 35ndash47

14 Korte J Alber M Trujillo C M Syson K Koliwer-Brandl HDeenen R Kohrer K DeJesusM AHartman T JacobsWR JrBornemann S Ioerger T R Ehrt S and Kalscheuer R (2016)Trehalose-6-phosphate-mediated toxicity determines essentiality ofOtsB2 inMycobacterium tuberculosis in vitroand inmicePLoSPathog 12e1006043

15 Lapp D Patterson B W and Elbein A D (1971) Properties of atrehalosephosphate synthetase fromMycobacteriumsmegmatismdashactivationof theenzymebypolynucleotidesandotherpolyanions JBiolChem2464567ndash4579

16 Matula M Mitchell M and Elbein A D (1971) Partial purificationand properties of a highly specific trehalose phosphate phosphatasefromMycobacterium smegmatis J Bacteriol 107 217ndash222

17 Reinders A Burckert N Hohmann S Thevelein J M Boller TWiemken A and De Virgilio C (1997) Structural analysis of thesubunits of the trehalose-6-phosphate synthasephosphatase com-plex in Saccharomyces cerevisiae and their function during heat shockMol Microbiol 24 687ndash695

18 McDougall J Kaasen I and Stroslashm A R (1993) A yeast gene fortrehalose-6-phosphate synthase and its complementation of anEscherichia coli otsA mutant FEMS Microbiol Lett 107 25ndash30

19 ParkHCBae YUCho SDKimSAMoon J YHaKCKimDW Lee K Jeong Y K KwackDOHeo J S Lee YG and JooW H (2007) Toluene-induced accumulation of trehalose by Pseudo-monas sp BCNU 106 through the expression of otsA and otsB ho-mologues Lett Appl Microbiol 44 50ndash55

20 Altschul S F Madden T L Schaffer A A Zhang J Zhang ZMiller W and Lipman D J (1997) Gapped BLAST and PSI-BLASTa new generation of protein database search programs Nucleic AcidsRes 25 3389ndash3402

21 Bryson K McGuffin L J Marsden R L Ward J J Sodhi J S andJones D T (2005) Protein structure prediction servers at UniversityCollege London Nucleic Acids Res 33(Web Server) W36ndashW38

22 Wang C K Broder U Weeratunga S K Gasser R B Loukas Aand Hofmann A (2012) SBAL a practical tool to generate and editstructure-based amino acid sequence alignments Bioinformatics 281026ndash1027

23 Cross M Lepage R Rajan S Biberacher S Young N D KimB N Coster M J Gasser R B Kim J S and Hofmann A (2017)Probing function and structure of trehalose-6-phosphate phospha-tases from pathogenic organisms suggests distinct molecular group-ings FASEB J 31 920ndash926

24 Weeratunga S Hu N J Simon A andHofmann A (2012) SDARa practical tool for graphical analysis of two-dimensional data BMCBioinformatics 13 201

25 KellerP andDunnP J (1982)Arsendescloizite anewmineral fromTsumebMineral Rec 13 155ndash157

26 KabschW (2010)XDSActa Crystallogr D Biol Crystallogr 66 125ndash13227 Winn M D Ballard C C Cowtan K D Dodson E J Emsley P

Evans P R Keegan R M Krissinel E B Leslie A G McCoy AMcNicholas S J Murshudov G N Pannu N S Potterton E APowell H R Read R J Vagin A andWilson K S (2011)Overviewof the CCP4 suite and current developments Acta Crystallogr D BiolCrystallogr 67 235ndash242

28 Panjikar S Parthasarathy V Lamzin V SWeissM S andTuckerP A (2005) Auto-rickshaw an automated crystal structure de-termination platform as an efficient tool for the validation of anX-raydiffraction experiment Acta Crystallogr D Biol Crystallogr 61 449ndash457

29 Sheldrick G M (2010) Experimental phasing with SHELXCDEcombining chain tracing with density modification Acta Crystallogr DBiol Crystallogr 66 479ndash485

30 Hao Q (2004) ABS a program to determine absolute configurationand evaluate anomalous scatterer substructure J Appl Cryst 37498ndash499

31 Pannu N S McCoy A J and Read R J (2003) Application of thecomplex multivariate normal distribution to crystallographicmethods with insights into multiple isomorphous replacementphasing Acta Crystallogr D Biol Crystallogr 59 1801ndash1808

32 Terwilliger T C (2000) Maximum-likelihood density modificationActa Crystallogr D Biol Crystallogr 56 965ndash972

33 Morris R J Zwart P H Cohen S Fernandez F J Kakaris MKirillova O Vonrhein C Perrakis A and Lamzin V S (2004)Breaking good resolutions with ARPwARP J Synchrotron Radiat 1156ndash59

34 Painter J and Merritt E A (2006) TLSMD web server for thegeneration of multi-group TLS models J Appl Cryst 39 109ndash111

35 Emsley P LohkampB ScottWG andCowtanK (2010) Featuresand development of Coot Acta Crystallogr D Biol Crystallogr 66486ndash501

36 Jones T A Zou J Y Cowan S W and Kjeldgaard M (1991)Improved methods for building protein models in electron densitymaps and the location of errors in thesemodelsActa Crystallogr A 47110ndash119

37 Adams P D Afonine P V Bunkoczi G Chen V B Davis I WEchols N Headd J J Hung L W Kapral G J Grosse-KunstleveR W McCoy A J Moriarty N W Oeffner R Read R JRichardsonDC Richardson J S TerwilligerTC andZwartPH(2010) PHENIX a comprehensive Python-based system for macro-molecular structure solution Acta Crystallogr D Biol Crystallogr 66213ndash221

38 Krissinel E and Henrick K (2007) Inference of macromolecularassemblies from crystalline state J Mol Biol 372 774ndash797

39 Van Der Spoel D Lindahl E Hess B Groenhof G Mark A Eand Berendsen H J (2005) GROMACS fast flexible and freeJ Comput Chem 26 1701ndash1718

40 DardenT YorkD andPedersen L (1993) ParticlemeshEwald anNtimeslog(N) method for Ewald sums in large systems J Chem Phys 9810089ndash10092

41 Bussi G Donadio D and ParrinelloM (2007)Canonical samplingthrough velocity rescaling J Chem Phys 126 014101

42 Parrinello M and Rahman A (1981) Polymorphic transitions insingle crystals a new molecular dynamics method J Appl Phys 527182ndash7190

43 Hess B BekkerH BerendsenH JC andFraaije J (1997)LINCSa linear constraint solver for molecular simulations J Comput Chem18 1463ndash1472


44 The Grace Team (2015) Grace 5125 Userrsquos GuideCanonical GroupLondon UKAQ12

45 Farelli J D Galvin B D Li Z Liu C Aono M Garland MHallett O E Causey T B Ali-Reynolds A Saltzberg D J CarlowC K Dunaway-Mariano D and Allen K N (2014) Structure of thetrehalose-6-phosphate phosphatase from Brugia malayi reveals keydesign principles for anthelmintic drugs PLoS Pathog 10 e1004245

46 R Core Team (2016) R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing Vienna Austria

47 Burroughs AM Allen K N Dunaway-MarianoD and Aravind L(2006) Evolutionary genomics of the HAD superfamilyunderstanding the structural adaptations and catalytic diversity in asuperfamily of phosphoesterases and allied enzymes J Mol Biol 3611003ndash1034

48 Allen K N and Dunaway-Mariano D (2009) Markers of fitness in asuccessful enzyme superfamily Curr Opin Struct Biol 19 658ndash665

49 Seifried A Schultz J and Gohla A (2013) Human HADphosphatases structure mechanism and roles in health anddisease FEBS J 280 549ndash571

50 Harris T K and Turner G J (2002) Structural basis of perturbedpKa values of catalytic groups in enzyme active sites IUBMB Life 5385ndash98

51 Di Nardo G Breitner M Bandino A Ghosh D Jennings G KHackett J C and Gilardi G (2015) Evidence for an elevatedaspartate pKa in the active site of human aromatase J Biol Chem 2901186ndash1196

52 Cross M Rajan S Chekaiban J Saunders J Hamilton C KimJ S Coster M J Gasser R B and Hofmann A (2017) Enzymecharacteristics of pathogen-specific trehalose-6-phosphate phospha-tases Sci Rep 7 2015

53 Kung V L Ozer E A and Hauser A R (2010) The accessorygenome of Pseudomonas aeruginosa Microbiol Mol Biol Rev 74621ndash641

54 QiuX Kulasekara B R andLory S (2009)Role of horizontal genetransfer in the evolution of Pseudomonas aeruginosa virulence InGenome Dynamics (deReuse H and andBereswill S eds) vol 6 pp126ndash139 Karger Basel Switzerland

55 Juhas M van der Meer J R Gaillard M Harding R M HoodD W and Crook D W (2009) Genomic islands tools of bacterialhorizontal gene transfer and evolution FEMS Microbiol Rev 33376ndash393

56 Tardif G and Grant R B (1982) Transfer of IncN plasmids toPseudomonas aeruginosa Antimicrob Agents Chemother 22 142ndash144

57 Gaballa A Abeysinghe P D Urich G Matthijs S De Greve HCornelis P and Koedam N (1997) Trehalose induces antagonismtowards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400Appl Environ Microbiol 63 4340ndash4345

58 Matthijs S Koedam N Cornelis P and De Greve H (2000) Thetrehalose operon of Pseudomonas fluorescens ATCC 17400 ResMicrobiol 151 845ndash851

59 Janin J (1997) Specific versus non-specific contacts in protein crys-tals Nat Struct Biol 4 973ndash974

60 Benkovic S J and Hammes-Schiffer S (2003) A perspective onenzyme catalysis Science 301 1196ndash1202

61 SimopoulosTT and JencksWP (1994)Alkalinephosphatase is analmost perfect enzyme Biochemistry 33 10375ndash10380

62 Stratton JR Pelton JG andKirsch J F (2001)Anovel engineeredsubtilisin BPN9 lacking a low-barrier hydrogen bond in the catalytictriad Biochemistry 40 10411ndash10416

63 Bennett B D Kimball E H Gao M Osterhout R VanDien S Jand Rabinowitz J D (2009) Absolutemetabolite concentrations andimpliedenzymeactive siteoccupancy inEscherichia coliNat ChemBiol5 593ndash599

64 Bar-Even A Noor E Savir Y Liebermeister W Davidi D TawfikD S and Milo R (2011) The moderately efficient enzymeevolutionary and physicochemical trends shaping enzymeparameters Biochemistry 50 4402ndash4410

65 Liu C Dunaway-Mariano D and Mariano P S (2017) Rationaldesign of reversible inhibitors for trehalose 6-phosphatephosphatases Eur J Med Chem 128 274ndash286

66 Edavana VK Pastuszak I Carroll JD Thampi P AbrahamECand Elbein A D (2004) Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis comparison tothe enzyme fromMycobacterium smegmatis Arch Biochem Biophys 426250ndash257

67 Guo F B Xiong L Zhang K Y Dong C Zhang F Z and WooP C (2017) Identification and analysis of genomic islands in

Burkholderia cenocepacia AU 1054 with emphasis on pathogenicityislands BMC Microbiol 17 73

68 Nzula S Vandamme P and Govan J R (2000) Sensitivity of theBurkholderia cepacia complex and Pseudomonas aeruginosa totransducing bacteriophages FEMS Immunol Med Microbiol 28307ndash312

69 Weaver V B and Kolter R (2004) Burkholderia spp alterPseudomonas aeruginosa physiology through iron sequestrationJ Bacteriol 186 2376ndash2384

70 Eberl L and Tummler B (2004) Pseudomonas aeruginosa andBurkholderia cepacia in cystic fibrosis genome evolution interactionsand adaptation Int J Med Microbiol 294 123ndash131

71 Vanaporn M Sarkar-Tyson M Kovacs-Simon A Ireland P MPumirat P Korbsrisate S Titball R W and Butt A (2017)Trehalase plays a role in macrophage colonization and virulence ofBurkholderia pseudomallei in insect and mammalian hosts Virulence 830ndash40

72 Schwarz S and Van Dijck P (2017) Trehalose metabolism a sweetspot for Burkholderia pseudomallei virulence Virulence 8 5ndash7

73 BlazquezM A Lagunas R Gancedo C andGancedo JM (1993)Trehalose-6-phosphate anewregulatorof yeast glycolysis that inhibitshexokinases FEBS Lett 329 51ndash54

74 Eastmond P J vanDijken A J SpielmanM Kerr A Tissier A FDickinson H G Jones J D Smeekens S C and Graham I A(2002) Trehalose-6-phosphate synthase 1 which catalyses the firststep in trehalose synthesis is essential for Arabidopsis embryo matu-ration Plant J 29 225ndash235

75 Thevelein J M andHohmann S (1995) Trehalose synthase guardto the gate of glycolysis in yeast Trends Biochem Sci 20 3ndash10

76 Puttikamonkul SWillger SDGrahlN Perfect JRMovahedNBothner B Park S Paderu P Perlin D S and Cramer R A Jr(2010) Trehalose 6-phosphate phosphatase is required for cell wallintegrity and fungal virulence but not trehalose biosynthesis in thehuman fungal pathogen Aspergillus fumigatus Mol Microbiol 77891ndash911

77 YadavU P IvakovA Feil RDuanGYWaltherDGiavalisco PPiques M Carillo P Hubberten H M Stitt M and Lunn J E(2014) The sucrose-trehalose 6-phosphate (Tre6P) nexus specificityand mechanisms of sucrose signalling by Tre6P J Exp Bot 651051ndash1068

78 Zhang Y Primavesi L F Jhurreea D Andralojc P J MitchellR A C Powers S J Schluepmann H Delatte T Wingler A andPaul M J (2009) Inhibition of SNF1-related protein kinase1 activityand regulation ofmetabolic pathways by trehalose-6-phosphate PlantPhysiol 149 1860ndash1871

79 Deroover S Ghillebert R Broeckx T Winderickx J and RollandF (2016) Trehalose-6-phosphate synthesis controls yeast gluconeo-genesis downstream and independent of SNF1 FEMS Yeast Res 16fow036

80 Nunes C Primavesi L F Patel M K Martinez-Barajas E PowersS J Sagar R Fevereiro P S Davis B G and Paul M J (2013)Inhibition of SnRK1 by metabolites tissue-dependent effects andcooperative inhibition by glucose 1-phosphate in combination withtrehalose 6-phosphate Plant Physiol Biochem 63 89ndash98

81 Claridge C A (1953) The Metabolism of Glucose by Pseudomonasaeruginosa Iowa State University Ames IA USA

82 Campbell J J Hoggla and Strasdine G A (1962) Enzymedistribution in Pseudomonas aeruginosa J Bacteriol 83 1155ndash1160

83 Kuser P R Krauchenco S Antunes O A C and Polikarpov I(2000) The high resolution crystal structure of yeast hexokinase PIIwith the correct primary sequence provides new insights into itsmechanism of action J Biol Chem 275 20814ndash20821

84 Kuser P Cupri F Bleicher L and Polikarpov I (2008) Crystalstructure of yeast hexokinase PI in complex with glucose a classicalldquoinduced fitrdquo example revised Proteins 72 731ndash740

85 Schneider T R and Sheldrick G M (2002) Substructure solutionwith SHELXD Acta Crystallogr D Biol Crystallogr 58 1772ndash1779

86 Brunger A T (1993) Assessment of phase accuracy by crossvalidation the free R value Methods and applications ActaCrystallogr D Biol Crystallogr 49 24ndash36

87 Inkscape Board of Developers (2017) Inkscape Userrsquos Guide FreeSoftware Foundation Boston MA USA

88 DeLanoWL (2002)ThePyMOLmolecular graphics systemAvailableat AQ13httpwwwpymolorg

Received for publication March 15 2018Accepted for publication April 16 2018


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AQ1mdash Per editorial policy color figures are converted to black and white if color used only foraesthetic purposes does not add to the content or data therefore if applicable figures wereconverted to black and white


AQ3mdash Please confirm or correct the suggested short title TPP OF PSEUDOMONAS AERUGINOSA

AQ4mdash Suppliers may have been edited to reflect mergers acquisitions and changes in headquarters

AQ5mdash Supplemental Table S7 was renumbered as Supplemental Table S1 per Journal stylebecause it is the only Supplemental table called out Similarly in the next paragraphSupplemental Information S3 has been relabeled as Supplemental Data S1

AQ6mdash Please confirm the resolution of TPP022 was 37 angstrom as edited

AQ7mdash Tables must be called out consecutively please cite Table 1 before this point where Table 2is called out delete this citation or mark the tables for renumbering

AQ8mdash The locants were removed from the Supplemental Figure S1 citation per Journal policybecause only some panels were called out in text

AQ9mdash Refs 85ndash88 were not cited in the text We have cited them in the last sentence Pleasereview the placement of the citation

AQ10mdash A required conflict of interest statement was added to the Acknowledgments Pleaseconfirm if correct

AQ11mdash If ldquoDAAD RISErdquo and ldquoPROMOSrdquo are acronyms please spell out

AQ12mdash Please confirm references 44 and 87 as edited If software rather than manuals are beingreferenced they need to become in-text references in the Materials and Methods sectionand all subsequent references must be renumbered

AQ13mdash Editor Is an access date still required for websites

AQ14mdash Is the addition of 7 to the legend of Fig 7 correct

AQ15mdash Table 2 headings have been reformatted to conform to journal style PDB accessionnumber has been moved to footnotes Please confirmcorrect first column heading

AUTHOR QUERIES

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references to software have been inserted in the textfigure captions and the relevant entries in the reference list have been removed References 85 and 86 have been renumbered as 45 and 46 in accordance with their appearance in the article
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1 Update to Adobe Acrobat Reader DCThe screen images in this document were captured on a Windows PC running Adobe Acrobat Reader DC Upgrading to the newest version is not always necessary but it is preferable and these instructions apply only to Adobe Acrobat Reader DC You can also create annotations using any version of Adobe Acrobat Adobe Acrobat Reader DC can be downloaded at no cost from httpgetadobecomreader

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Annotating PDFs using Adobe Acrobat Reader DCVersion 17 June 27 2016

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8 Still have questionsTry viewing our brief training video at httpsauthorcenterdartmouthjournalscomArticlePdfAnnotation

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0892-6638180032-0001 copy FASEB 1

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Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





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0892-6638180032-0001 copy FASEB 1

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Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



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Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

Author Instructions








Manuscript No

Title












1 Print Name

Signature Date

2 Print Name

Signature Date

3 Print Name

Signature Date

4 Print Name

Signature Date

5 Print Name

Signature Date

6 Print Name

Signature Date

7 Print Name

Signature Date

8 Print Name

Signature Date

9 Print Name

Signature Date

10 Print Name

Signature Date


THE















0892-6638180032-0001 copy FASEB 1

ah
Sticky Note









COLOR



ah
Inserted Text
(see Table 1)
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Highlight
ah
Sticky Note
ah
Inserted Text
)






Crystallization








ah
Cross-Out
ah
Replacement Text
1
ah
Inserted Text
to
ah
Cross-Out
ah
Cross-Out


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok



Manuscript No

Title












1 Print Name

Signature Date

2 Print Name

Signature Date

3 Print Name

Signature Date

4 Print Name

Signature Date

5 Print Name

Signature Date

6 Print Name

Signature Date

7 Print Name

Signature Date

8 Print Name

Signature Date

9 Print Name

Signature Date

10 Print Name

Signature Date


THE















0892-6638180032-0001 copy FASEB 1

ah
Sticky Note









COLOR



ah
Inserted Text
(see Table 1)
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Highlight
ah
Sticky Note
ah
Inserted Text
)






Crystallization








ah
Cross-Out
ah
Replacement Text
1
ah
Inserted Text
to
ah
Cross-Out
ah
Cross-Out


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

THE















0892-6638180032-0001 copy FASEB 1

ah
Sticky Note









COLOR



ah
Inserted Text
(see Table 1)
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Highlight
ah
Sticky Note
ah
Inserted Text
)






Crystallization








ah
Cross-Out
ah
Replacement Text
1
ah
Inserted Text
to
ah
Cross-Out
ah
Cross-Out


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok









COLOR



ah
Inserted Text
(see Table 1)
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Highlight
ah
Sticky Note
ah
Inserted Text
)






Crystallization








ah
Cross-Out
ah
Replacement Text
1
ah
Inserted Text
to
ah
Cross-Out
ah
Cross-Out


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok






Crystallization








ah
Cross-Out
ah
Replacement Text
1
ah
Inserted Text
to
ah
Cross-Out
ah
Cross-Out


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok


Chemicals







RESULTS














ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
also






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok






Se-Met

TPP022 TPP023g




ab 0057 (0201) 0101 (0527)Rmeas

a 0068 (0234) 0118 (0604)Rpim





22 1 Mg2+ 1 CO322 1 Mg2+ 1 CO3

22





af 0218 (0301)



ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
4
ah
Sticky Note
109
ah
Sticky Note
105
ah
Cross-Out
ah
Cross-Out






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok






COLOR



ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
one
ah
Cross-Out
ah
Replacement Text
-helical

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

















ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
ah
Cross-Out
ah
Replacement Text
and wraps
ah
Cross-Out
ah
Replacement Text
3
ah
Cross-Out
ah
Replacement Text
2
ah
Cross-Out
ah
Replacement Text
5
ah
Cross-Out
ah
Replacement Text
3




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok




COLOR


COLOR



ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

DISCUSSION







COLOR







ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Cross-Out
ah
Replacement Text
Data S1
ah
Sticky Note
ah
Sticky Note
ah
Cross-Out
ah
Replacement Text
one







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok







COLOR


COLOR



ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
httpswwwinkscapeorg
ah
Cross-Out
ah
Replacement Text
httpspymolorg
ah
Cross-Out
ah
Replacement Text
httpsinkscapeorg






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok






CONCLUSIONS









ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Inserted Text
ah
Sticky Note

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

REFERENCES





























































































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

















































ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
ah
Inserted Text
4
ah
Cross-Out
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Replacement Text
4
ah
Cross-Out
ah
Cross-Out
ah
Cross-Out
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok
















AUTHOR QUERIES


ah
Sticky Note
ok
ah
Sticky Note
ok na
ah
Sticky Note
ok na
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
done
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ah
Sticky Note
ok
ah
Sticky Note
ok

Documents

1 Dear FASEB Journal Author