EF5 Is the High-Affinity Mg2+ Site in ALG‑2John J. Tanner,†,‡ Benjamin B. Frey,† Travis Pemberton,‡ and Michael T. Henzl*,†

†Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, United States‡Department of Chemistry, University of Missouri, Columbia, Missouri 65211, United States

ABSTRACT: The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) hasbeen implicated in several important physiological processes, including endoplasmicreticulum−Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 wasrecently shown to harbor a metal ion-binding site with a high affinity for Mg2+ and alow affinity for Ca2+. We herein present the X-ray structure of Mg2+-bound ALG-2des23wt. Although the Cα trace is nearly indistinguishable from that of the Ca2+-freeprotein, the orientation of the C-terminal helix differs in the two structures. Consistentwith that observation, replacement of the +x ligand in EF5, D169, with alanineeliminates high-affinity Mg2+ binding. It also eliminates the low-affinity Ca2+ site andlowers the affinity of the remaining Ca2+-binding sites, EF3 and EF1. The coordination environment in EF5 approaches idealMg2+ octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (−y), and twowater molecules (−x, −z), may offer a recipe for a high-affinity, high-selectivity Mg2+-binding site. Sequence data for other PEFproteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg2+-bindingsite. In Mg2+-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium groupof K137 in the opposing subunit, suggesting that Mg2+ binding could have an impact on dimerization. Interestingly, EF1 and EF3are also occupied in the crystal, despite having modest affinity for Mg2+. The results of a calorimetry-based analysis indicate thattheir Mg2+ binding constants are 2 orders of magnitude lower than that determined for EF5.

EF-hand proteins1−6 participate in numerous eukaryotic signaltransduction pathways.7−9 The “EF-hand” motif includes ametal ion-binding loop and flanking helical segments, thespatial orientation of which can be suggested by the right hand.Although the motifs typically occur as tandem pairs, the dimericpenta-EF-hand (PEF) proteins10 possess an unpaired C-terminal EF-hand, which serves as a dimerization domain.The PEF protein ALG-2 (short for apoptosis-linked gene

2)11−13 was discovered during a search for gene products thatconferred protection from ligand-induced apoptosis in a T-cellhybridoma.14 Highly conserved, ALG-2 displays a broad tissuedistribution14,15 and is found in the nucleus and cytoplasm.16,17

Two isoforms are expressed in vivo.13,15 The more abundantone, often denoted ALG-2wt, includes 191 residues. Roughlyone-third of the time, an alternative splicing event excises thecodons for G121 and F122, yielding ALG-2ΔGF122. The twoisoforms can form a heterodimeric complex.15

Ca2+ binding triggers exposure of apolar surface, forinteraction with target proteins. ALG-2 is proposed to functionas an adaptor molecule, bridging unrelated proteins orstabilizing weak protein−protein complexes. The addition ofCa2+ to cell lysates promotes translocation of ALG-2 to themembranous fraction, implying association with membrane-localized proteins.13,18 A majority of putative biological targetsidentified to date harbor specific proline-rich regions (PRRs)known as ALG-2-binding motifs (ABMs). The ALG-2 isoformsexhibit distinct target protein specificities. Whereas ALG-2wt

associates with either ABM-1 (PPYPXXPGYP) or ABM-2(PXPGF) motifs, ALG-2ΔGF122 interacts exclusively with ABM-2.15,19,20

Although its name would suggest otherwise, ALG-2 activitycan promote either cell death or cell proliferation.11 Theinfluence on cell survival pathways is probably an indirectconsequence of its involvement in several important physio-logical processes. These include (1) endoplasmic reticulum(ER)−Golgi vesicular transport, through interactions withSec31a and annexin A11,21−27 (2) endosomal biogenesis andtransport, via interactions with Alix/AIP128,29 and Tsg101,30

and (3) cell membrane repair,31 likewise via interaction withAlix/AIP1. Curiously, elimination of the ALG-2 gene has noapparent physiological impact: ALG-2−/− mice developnormally and display no obvious immune defect.32

45Ca2+ flow-dialysis measurements revealed that both ALG-2isoforms possess two high-affinity Ca2+ sites and one low-affinity site.15 EF1 and EF3 are the high-affinity sites,33,34 andEF5 is the low-affinity site. A one-residue insertion in the EF5-binding loop prevents the glutamyl residue at the C-terminalend of the loop from serving as the canonical bidentate Ca2+

ligand. Inclusion of 2 mM Mg2+ in the flow-dialysis assays hadno discernible impact on Ca2+ affinity in either isoform,suggesting that the ALG-2 Ca2+-binding sites are specific forCa2+. However, a recent ITC study convincingly demonstratedthat both ALG-2 isoforms possess a high-affinity Mg2+ site.35

We herein describe the X-ray structure of ALG-2des23wt

crystallized in the presence of 1.0 mM Mg2+. The tertiarystructure of the Mg2+-bound molecule closely resembles that of

Received: June 12, 2016Revised: August 18, 2016Published: August 19, 2016

Article

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© 2016 American Chemical Society 5128 DOI: 10.1021/acs.biochem.6b00596Biochemistry 2016, 55, 5128−5141

the apoprotein structure reported previously.36 The mostprominent structural difference appears in the C-terminal helix,suggesting that EF5 is the high-affinity Mg2+ site. Consistentwith that hypothesis, replacement of D169 (full-length ALG-2wt

numbering system) with alanine abolishes high-affinity Mg2+

binding, eliminates the low-affinity Ca2+ site, and attenuates theCa2+ affinity at the two remaining sites. The structural changesthat accompany the binding of Mg2+ in EF5 could potentiallyinfluence the kinetics and/or energetics of dimerization.Because the dimeric structure of ALG-2 is integral to itsproposed adaptor function, Mg2+ binding could modulate ALG-2 activity.EF1 and EF3 are also occupied by Mg2+ in the crystal. The

unanticipated presence of Mg2+ in these low-affinity sitesprompted an effort to obtain estimates for their Mg2+ bindingparameters, employing an ITC-based analysis of competitiveCa2+ and Mg2+ binding.

■ MATERIALS AND METHODSReagents. These items were purchased from Fisher

Scientific: ampicillin, CaCl2·H2O, MgCl2·2H2O, Na2EDTA·2H2O, glycerol, Hepes, KCl, lysozyme, and 2-propanol. Thefollowing were obtained from Sigma-Aldrich: chloramphenicol,EGTA, NTA, 1.0 M Tris buffer solutions, Tween 20, and high-purity (>99.5%) urea. IPTG was obtained from GoldBiotechnology. LB broth capsules were purchased fromResearch Products International.Mutagenesis. The coding sequence for ALG-2des23wt, an

ALG-2wt construct lacking residues 1−23, had previously beencloned into pET11. Asp 169 (full-length ALG-2wt sequencenumbering) was replaced with alanine using the QuikChangemutagenesis kit (Agilent), employing oligonucleotides pur-chased from Integrated DNA Technologies (Coralville, IA).The fidelity of the mutated sequence was confirmed byautomated DNA sequencing at the University of Missouri DNACore Facility.Protein Expression and Isolation. Rosetta 2(DE3) cells

(Novagen) harboring the construct of interest (ALG-2des23wt

or ALG-2des23wt/D169A) were incubated at 37 °C in LB brothcontaining ampicillin (100 μg/mL) and chloramphenicol (30μg/mL). When the absorbance at 600 nm reached 0.6, the 1 Lcultures were chilled to approximately 20 °C in an ice−waterbath. IPTG was added to a final concentration of 0.25 mM, andthe cultures were maintained at 23 °C while being shaken foran additional 16 h. The bacteria were collected bycentrifugation, resuspended in 20 mM Hepes (pH 7.4), andlysed by being treated with lysozyme and extrusion from aFrench pressure cell. The proteins were purified to apparenthomogeneity, as assessed by SDS−PAGE, employing theprocedure described by Lo et al.33

Crystallization. Crystals of ALG-2des23wt were grown at 4°C in hanging drops, using 24-well VDX plates (HamptonResearch). The precipitant contained 2-propanol [29−32% (v/v)], 0.10 M Tris-HCl (pH 7.4, 25 °C), 1.0 mM EGTA, and 1.0mM Mg2+. The inclusion of EGTA prevented binding of(contaminating) Ca2+ to the EF-hand motifs. The drops wereprepared by combining 2.0 μL of ALG-2des23wt [5.0 mg/mL in10 mM Hepes-KOH (pH 7.4)] with 2.0 μL of the precipitantsolution. The crystals, which appeared in 2−4 days, werepulverized and used to seed additional wells, resulting in largercrystals. The latter were prepared for low-temperature datacollection by being transferred to a cryobuffer (precipitantcontaining 25% glycerol) at 4 °C. The cryoprotected crystals

were harvested with loops and flash-cooled by rapid immersionin liquid nitrogen.

X-ray Diffraction Data Collection and Processing. X-ray diffraction data were collected at beamline 4.2.2 of theAdvanced Light Source, using a CMOS-based Taurus-1detector in shutterless mode. The data set used for refinement,collected at a detector distance of 210 mm, consisted of 1800frames spanning 180°. The total exposure time was 360 s. Datawere integrated and scaled with XDS.37 Intensities were mergedand converted to amplitudes with Aimless.38 ALG-2des23wt

crystallized in space group P21212, with the following unit celldimensions: a = 76.5 Å, b = 48.5 Å, and c = 54.3 Å. Because thedata were initially processed assuming a < b < c, the standardconvention for primitive orthorhombic space groups, theREINDEX module of ccp4i39 was used to reindex thereflections so that the 54.3 Å axis corresponds to the c-axis,i.e., transformation of (h,k,l) to (l,h,k).

Refinement. Refinement with PHENIX40 was initiated withthe coordinates from a structure of N-terminally truncatedhuman ALG-2 [Protein Data Bank (PDB) entry 2ZND] thatlikewise crystallized in P21212, the same cell used here formouse ALG-2des23wt. The first round of refinement used rigid-body refinement and simulated annealing. The B-factor modelconsisted of one TLS group encompassing the entire proteinchain and an isotropic B-factor for each non-hydrogen atom. Allatoms have an occupancy of 1.0.

Estimation of the Uncertainties of Bond Lengths andBond Angles. The uncertainties in the bond lengths andangles for the Mg2+ sites were estimated from structures refinedagainst six diffraction data sets collected from four crystals(Table 5). The six data sets included the one used to generatethe deposited structure (Table 1) and five others having high-resolution limits between 1.75 and 1.80 Å (Table 5). For eachdata set, the refined 1.72 Å resolution structure (see Table 1)was used to initiate 10 independent simulated annealingrefinement calculations. Prior to each refinement calculation,the coordinates of each atom were perturbed by applying arandom shift, using the “shake” option of phenix.pdbtools(mean displacement parameter set to 0.4 Å). The 60 resultingstructures were used to estimate standard deviations for thebond lengths and angles.

Sedimentation Velocity Analyses. ALG-2des23wt and theD169A variant were analyzed by sedimentation velocity at 20°C in a Beckman XL-I Optima analytical ultracentrifuge.Aliquots of protein (400 μL) and buffer (430 μL), in 0.15 MKCl, 0.025 M Hepes, and 1.0 mM EDTA (pH 7.4), wereloaded into the sample and solvent chambers, respectively, of asedimentation velocity cell, equipped with a charcoal-Epondual-sector centerpiece. The absorbance of the sample was 0.80at 280 nm, in a 1.0 cm cuvette. Assuming a molar absorptivityof 36100 M−1 cm−1, the nominal protein (monomer)concentration was 22 μM. Following temperature equilibration,the sample was centrifuged at 30000 rpm, with data beingacquired continuously until 300 radial scans had been collected.The resulting data set was analyzed globally with Sedfit toobtain the sedimentation coefficient, c(s), and molecularweight, c(M), distributions.

Urea Denaturation Studies. The impact of Mg2+ on theapparent conformational stability was evaluated by titratingsamples of ALG-2des23wt and D169A with urea at 25 °C, in theabsence or presence of 1.0 mM Mg2+. Unfolding was monitoredby circular dichroism in a 1.0 cm cuvette, employing an AVIV202 spectrometer, equipped with a Hamilton Microlab 500

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automated titrator. The protein samples (3.0 μM) and the 10.0M urea titrant were prepared in 0.15 M KCl, 0.010 Mpotassium phosphate, and 0.5 mM EGTA (pH 7.4) in thepresence or absence of 1.0 mM Mg2+. The titrant concentrationwas confirmed by refractometry. Titrations were conducted in aconstant volume of 2.0 mL. Following each addition of titrant,the sample was stirred for 60 s, and the ellipticity at 222 nm wasthen measured for 30 s.Isothermal Titration Calorimetry. All ITC data were

collected at 25 °C with a VP-ITC instrument (MalvernInstruments). The experiments were conducted in 0.15 M KCl,0.025 M Hepes (pH 7.4), and 0.5% (v/v) Tween 20. Asdescribed previously,35 because Tween 20 is susceptible tohydrolysis, the detergent was added to the samples just prior tothe titration. Divalent metal ions were removed from theprotein preparations before ITC analysis by passage overEDTA-agarose.41,42 The residual Ca2+ content, measured byflame atomic absorption at 422.7 nm, was <0.02 equiv. Thebuffer solutions used to prepare the titrants and to rinse thesample cell prior to sample loading were treated similarly.Analysis of D169A Ca2+ Binding. The Ca2+ binding

constants and enthalpies for the D169A variant were estimatedby ITC. Aliquots of the protein were titrated with Ca2+ in the

absence and presence of chelators (EDTA, EGTA, and NTA).The resulting data were analyzed globally, using the bindingparameters for the small-molecule chelators reported pre-viously.35

As described elsewhere,42,43 the strategy used to calculate theheat associated with the ith titrant injection involves estimationof the free divalent ion concentration(s), calculation of thecumulative binding enthalpy after the ith injection, andsubtraction of the cumulative binding enthalpy associatedwith the previous injection.The D169A mutation abolishes high-affinity Mg2+ binding.

Because it also eliminates the low-affinity Ca2+ site, the Ca2+

binding behavior of the resulting protein can be described witha two-site Adair model. Estimation of the free Ca2+

concentration was achieved by minimizing the following FCfunction, employing a bisection strategy:

= +

++ +

−

+

+ +

+ ++

⎛⎝⎜⎜

⎞⎠⎟⎟

n

K K K

K K K

FC [Ca ] [P]

[Ca ] 2 [Ca ]

1 [Ca ] [Ca ][Ca ]

2t

Ca,12

Ca,2 Ca,12 2

Ca,12

Ca,2 Ca,12 2

2t

where KCa,1 and KCa,2 are stepwise macroscopic bindingconstants for the first and second Ca2+ binding events,respectively, [Ca2+]t and [P]t represent the total Ca2+ andprotein concentrations, respectively, and n is a stoichiometricfactor that allows for uncertainty in the protein concentration.In the presence of a low-molecular weight chelator (e.g.,

EGTA), FC assumes the following form:

= ++

++

+ +

−

++

+

+ +

+ +

+

⎛⎝⎜

⎞⎠⎟

⎛⎝⎜⎜

⎞⎠⎟⎟

KK

nK K K

K K K

FC [Ca ] [EGTA][Ca ]

1 [Ca ]

[P][Ca ] 2 [Ca ]

1 [Ca ] [Ca ]

[Ca ]

2t

EGTA2

EGTA2

tCa,1

2Ca,2 Ca,1

2 2

Ca,12

Ca,2 Ca,12 2

2t

where [EGTA]t is the total EGTA concentration and KEGTA isthe Ca2+ binding constant for EGTA.Given an estimate for [Ca2+], the cumulative binding heat

after the ith injection (Qi) is calculated with the followingequation, where ΔHCa,1 and ΔHCa,2 represent the molarenthalpy changes associated with the binding events:

=

Δ + Δ + Δ+ +

+ +

+ +

⎡⎣⎢⎢

⎤⎦⎥⎥

Q V n

H K H H K K

K K K

[P]

[Ca ] ( ) [Ca ]

1 [Ca ] [Ca ]

i o t

Ca,1 Ca,12

Ca,1 Ca,2 Ca,2 Ca,12 2

Ca,12

Ca,2 Ca,12 2

An estimate for the injection heat is obtained by taking thedifference between the cumulative heats associated with the iand i − 1 data points and adding a correction for the volume ofthe reaction mixture displaced by the ith injection (dV) and abaseline offset (BL):

= − ++

+−−

⎛⎝⎜

⎞⎠⎟q Q Q

VV

Q Qd2

BLi i ii i

1o

1

where Vo is the sample cell volume (1.41 mL).Global Analysis of Divalent Ion Binding by ALG-

2des23wt. Upon learning that all three Ca2+-binding sites inALG-2des23wt are occupied by Mg2+ in the crystal, we becameinterested in the Mg2+ affinities of the two low-affinity sites. Wehad previously estimated the binding constants and enthalpies

Table 1. Data Collection and Refinement Statisticsa

beamline ALS 4.2.2space group P21212unit cell parameters (Å) a = 76.5, b = 48.5, c = 54.3wavelength 1.0000resolution (Å) 54.35−1.72 (1.75−1.72)no. of observations 152354no. of unique reflections 22193Rmerge(I) 0.045 (1.014)Rmeas(I) 0.049 (1.103)Rpim(I) 0.018 (0.429)mean CC1/2 1.000 (0.770)mean I/σ 25.8 (1.6)completeness (%) 100.0 (100.0)multiplicity 6.9 (6.5)no. of protein residues 168no. of atoms/ions

protein 1398Mg2+ 3water 106

Rcryst 0.1797 (0.3082)Rfree

b 0.2075 (0.3279)rmsd for bond lengths (Å) 0.006rmsd for bond angles (deg) 0.694Ramachandran plotc (%)

favored 100.00outliers 0.00

Clashscore (percentile)c 0.72 (99th)MolProbity score (percentile)c 1.05 (100th)average B (Å2)

protein 29.6Mg2+ 26.9/26.5/27.2d

water 37.7coordinate error (Å)e 0.20PDB entry 5JJGaValues for the outer resolution shell of data are given in parentheses.bRandom 5% test set. cGenerated with MolProbity.51 dValues arelisted as EF1, EF3, EF5. eMaximum likelihood-based coordinate errorestimate from PHENIX refine.

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for the three Ca2+-binding sites in ALG-2des23wt and for thehigh-affinity Mg2+ site.35 Those values were obtained bymodeling titrations conducted in the absence and presence ofcompetitive chelators. To complete the analysis of divalent ionbinding by this protein, we have included titrations with Ca2+ inthe presence of Mg2+, titrations with Mg2+ in the presence ofCa2+, and simultaneous titrations with both metal ions (atseveral Mg2+:Ca2+ concentration ratios). The resulting datawere treated using a three-site Adair model.When [Ca2+] and [Mg2+] are both present, the ALG-

2des23wt monomer can, in principle, reside in 10 distinctligation states: P, CaP, Ca2P, Ca3P, MgP, Mg2P, Mg3P, CaMgP,Ca2MgP, and CaMg2P. Their respective contributions to thepartition function, PF, are shown below:

= + + +

+ +

+ +

+

+

+ +

+ + +

+ +

+ + +

+ +

K K K K K

K K K K

K K K K K

K K K

K K K

PF 1 [Ca ] [Ca ]

[Ca ] [Mg ] [Mg ]

[Mg ] CI [Ca ]

[Mg ] CI [Ca ] [Mg ]

CI [Ca ][Mg ]

Ca,12

Ca,2 Ca,12 2

Ca,3 Ca,2

Ca,12 3

Mg,12

Mg,2 Mg,12 2

Mg,3 Mg,2 Mg,12 3

Ca,1 Mg,1 CaMg2

2Ca,2 Ca,1 Mg,1 Ca Mg

2 2 2

Ca,1 Mg,1 Mg,2 CaMg2 2 2

2

2

In general, the statistical weights for each species equal theproduct of the respective association constants and the (free)metal ion concentrations raised to the appropriate powers. Theterms for the mixed complexes also include a “cooperativityindex” (CI), which measures the interaction between thebinding events for the two ions. Qualitative inspection ofcompetition data35 suggested that high-affinity Mg2+ binding isassociated with the low-affinity Ca2+ site, a hypothesisconfirmed in this work. Thus, in the CaMgP complex, thehighest-affinity Ca2+ site and the high-affinity Mg2+ site shouldboth be occupied. Accordingly, the statistical weight for thecomplex can be expressed as KCa,1KMg,1CICM[Ca

2+][Mg2+].Similarly, for the Ca2MgP complex, the two Ca2+ ions shouldoccupy the two higher-affinity sites, and the Mg2+ ion wouldoccupy the high-affinity site. The corresponding term in thep a r t i t i o n f u n c t i o n i s e q u a l t oKCa,1KCa,2KMg,1CIC2M[Ca

2+]2[Mg2+]. Finally, the term forCaMg2P was written as KCa,1KMg,2KMg,1CICM2[Ca

2+][Mg2+]2,assuming that the lone Ca2+ resides in the highest-affinity siteand the two bound Mg2+ ions reside in the high-affinity Mg2+

site (low-affinity Ca2+) and the intermediate-affinity Ca2+ site.In general, stepwise macroscopic binding constants do notcorrespond to specific binding sites.44 For ALG-2des23wt,however, the three binding sites exhibit distinctly different Ca2+

affinities. Under these circumstances, the stepwise macroscopicconstants approach the microscopic, site-specific values.It is necessary to estimate both the free Ca2+ and Mg2+

concentrations for modeling experiments in which both ionsare present. The FC function assumes the form

= + −+ +⎡⎣⎢

⎤⎦⎥nFC [Ca ] [P]

CBPF

[Ca ]2t

2t

where the Ca2+ binding function CB represents the amount ofbound metal ion, equal to

= + +

+

+ +

+ +

+ + +

+ +

+ +

K K K K K

K K K

K K K K K

K

CB [Ca ] 2 [Ca ] 3

[Ca ] CI [Ca ][Mg ]

2 CI [Ca ] [Mg ]

CI [Ca ][Mg ]

Ca,12

Ca,2 Ca,12 2

Ca,3 Ca,2

Ca,12 3

Ca,1 Mg,1 CaMg2 2

Ca,2 Ca,1 Mg,1 Ca Mg2 2 2

Ca,1 Mg,1

Mg,2 CaMg2 2 2

2

2

The corresponding FM function has a similar form

= + −+ +⎡⎣⎢

⎤⎦⎥nFM [Mg ] [P]

MBPF

[Mg ]2t

2t

where MB represents the bound Mg2+

= + +

+

+ +

+ +

+ + +

+ +

+ +

K K K K K

K K K

K K K K K

K

MB [Mg ] 2 [Mg ] 3

[Mg ] CI [Ca ][Mg ]

CI [Ca ] [Mg ] 2

CI [Ca ][Mg ]

Mg,12

Mg,2 Mg,12 2

Mg,3 Mg,2

Mg,12 3

Ca,1 Mg,1 CaMg2 2

Ca,2 Ca,1 Mg,1 Ca Mg2 2 2

Ca,1 Mg,1

Mg,2 CaMg2 2 2

2

2

The calculation of the cumulative heat parallels thatdescribed above for the simpler, two-site system. However,because of interactions between the bound ions, the heatassociated with formation of the mixed complexes includes aperturbation term, δH. For example, the contribution of theformation heat of the CaMgP species equals

δΔ + ΔΗ +V n H H[P] ( )o t Ca,1 Mg,1 CaMg

The corresponding term for Ca2Mg equals

δΔ + Δ + Δ +V n H H H H[P] ( )o t Ca,1 Ca,2 Mg,1 Ca Mg2

A Monte Carlo algorithm was employed for least-squaresminimization. The binding parameters, binding constants andenthalpies, for the three Ca2+-binding sites and the high-affinityMg2+ site were fixed at the previously determined values. Tooptimize the fits to the Ca2+/Mg2+ competition data, thefollowing parameters were varied: KMg,2, KMg,3, ΔHMg,2, ΔHMg,3,CICaMg, CICa2Mg, CICaMg2, δHCaMg, δHCa2Mg, and δHCaMg2. The CIand δH terms were initially set to 1.0 and 0, respectively.Additionally, the stoichiometries and baseline offsets for each ofthe 16 titrations were likewise allowed to float. Assuming auniform standard deviation for the injection heats of 0.35 μcal,the resulting reduced χ2 value was 1.95.For a given least-squares fit, departures of χ2 from the

observed minimum (χmin2) are statistically insignificant, up to

some limiting ratio given by the equation

χ χ = +p

F p P/ 1dof

( , dof, )2min

2

where p is the number of fitting parameters, dof the degrees offreedom, P the probability that the increase in χ2 is not merelythe result of random errors, and F(p, dof, P) the value of the F-statistic. In our case, p was set to 18. All of the associationconstants, binding enthalpies, cooperative indices, andperturbation enthalpies were varied, but the stoichiometriesand baseline values were fixed at their optimal values. The dofvalue was set to 650, half of the number of total data points(recognizing the minimal information content of baselineinjection heats at the end of the titrations). The F-statistic valuewas obtained using an online calculator.45 The limiting χ2/χmin

2

ratio obtained using this approach was 1.042, corresponding toa χmin

2 value of 2.032. Monte Carlo simulation was used to

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generate 5000 parameter sets yielding a χ2/χmin2 ratio within

0.5% of that χ2 limit. The uncertainty for each parameter isreported as half of the difference between the maximal andminimal parameter values.

■ RESULTSIn 30% 2-PrOH and 0.10 M Tris, containing 1.0 mM Mg2+,ALG-2des23wt formed crystals that diffracted to 1.72 Å. Theresulting structure bears a strong similarity to that of PDB entry2ZND,36 in which Na+ ions occupy EF1, EF3, and EF5. Asobserved for 2ZND, the Mg2+-bound protein likewisecrystallizes in space group P21212. The unit cell had thefollowing dimensions: a = 76.5 Å, b = 48.5 Å, and c = 54.3 Å.Additional data collection and processing statistics are listed inTable 1.A ribbon diagram of the Mg2+-bound protein is displayed in

Figure 1. Calorimetric analysis previously established that ALG-

2des23wt possesses a single high-affinity Mg2+-binding site.35

Interestingly, however, Mg2+ is present in EF1, EF3, and EF5.According to the CheckMyMetal server46 (http://csgid.org/csgid/metal_sites), the coordination geometry observed in allthree sites is most consistent with Mg2+. The coordinationenvironments for the three bound Mg2+ ions are displayed inFigure 2. The observed bond lengths and angles are listed inTable 2.As shown in Figure 2A, the Mg2+ ion in EF5 is coordinated at

the +x, +y, and +z positions by carboxylates (donated by D169,D171, and D173, respectively). The corresponding Mg−Odistances are 2.11, 2.00, and 2.11 Å, respectively. The main-chain carbonyl oxygen of W175 furnishes the −y ligand, with aMg−O bond length of 2.06 Å. Finally, water molecules occupythe −x and −z positions, with bond lengths of 2.23 and 1.99 Å,respectively. The FindGeo server47 (http://metalweb.cerm.unifi.it/tools/findgeo/) classified the coordination geometry asregular octahedral, with an rmsd equal to 0.20 Å, relative to anidealized Mg2+ geometry.The bound Mg2+ ion in EF3 is coordinated by just two

carboxylates, as shown in Figure 2B. D103 and D105 furnishthe +x and +y ligands, respectively, with Mg−O distances of2.09 and 2.04 Å, respectively. A serine hydroxyl (S107) iscoordinated at +z, with a bond length of 2.22 Å. The −y ligandis provided by the backbone carbonyl of M109, with a bondlength of 2.16 Å. Two water molecules complete the innercoordination sphere, with Mg−O distances of 2.31 Å (−x) and1.90 Å (−z). This coordination environment was likewise

classified as regular octahedral by FindGeo, with an rmsd valueof 0.22 Å.The Mg2+ in EF1 resides in a less regular environment

(Figure 2C). The inner coordination sphere is evidently formedfrom the carboxylates of D36 (+x) and D38 (+y), the hydroxylgroup from S40 (+z), the main-chain carbonyl of V42 (−y), awater molecule (−x), and the carboxylate of E47 (−z,monodentate). Fairly typical bond lengths, ranging from 2.00to 2.28 Å, are observed at +x, +y, −y, −x, and −z. However, thedistance between the bound Mg2+ and Oγ of S40 is 2.86 Å.Moreover, several of the bond angles depart significantly fromthe 90° and 180° values characteristic of octahedralcoordination. In fact, the FindGeo server classifies the EF1coordination environment as a distorted trigonal bipyramid,with an rmsd value of 0.524 Å.

Impact of Replacing Asp 169 with Alanine in ALG-2des23wt. In our previous ITC study,35 competition for Mg2+

in the EF-hand motifs of ALG-2des23wt was manifestedprimarily in the latter stages of Ca2+ titrations. Moreover,inclusion of 2 molar equivalents of Ca2+ had a minimal impacton the high-affinity Mg2+ binding event. These findings impliedthat the high-affinity Mg2+ site is associated with the low-affinityCa2+ site, EF5. The crystal structure of the Mg2+-bound proteinsupports that hypothesis. As discussed below, the mostapparent structural difference that accompanies Mg2+ binding

Figure 1. Ribbon diagram of Mg2+-bound ALG-2des23wt.The greenspheres represent the Mg2+ ions present in EF1, EF3, and EF5.

Figure 2. Mg2+ coordination environments in (A) EF5, (B) EF3, and(C) EF1.

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occurs at the C-terminus. Moreover, of the three occupied sites,EF5 exhibits the most regular Mg2+ coordination geometry,according to FindGeo.If EF5 is, in fact, the high-affinity Mg2+ site, then elimination

of a critical ligand in the coordination sphere should abolishhigh-affinity binding. Typically, mutation of the +x ligand issufficient to disrupt divalent ion binding in an EF-hand motif.Accordingly, we replaced D169 (wild-type ALG-2des23wt

numbering system) with alanine. Our analysis of the resultingD169A variant is described in the following paragraphs.Quaternary Structure. A sample of D169A was subjected

to sedimentation velocity analysis, to determine whether themutation in the EF5 loop impacted dimerization. The greenlines displayed in panels A and B of Figure 3 represent theresulting sedimentation coefficient, c(s), and molecular weight,c(M), distributions, respectively. For comparison, the corre-sponding distributions obtained for the wild-type protein arealso presented (red).The behaviors of the wild-type and variant proteins are

similar. Both yield symmetric c(s) distributions. The maximum

in the D169A distribution (3.10 S) is comparable to thatobserved for the wild-type protein (3.15 S), and the maxima inthe c(M) distributions are virtually identical, 40200 for ALG-2des23wt and 40500 for the variant. Evidently, D169A retains itsdimeric structure. The c(s) distribution obtained for the variantis slightly broader than the wild-type distribution, which mayreflect a minor difference in the conformational dynamics of thetwo proteins. Sedfit employs a weight-average frictional ratio( f/fo) to estimate a diffusion coefficient for the sedimentingprotein. If there is conformational heterogeneity, a singlefrictional value will not precisely capture the diffusion behaviorof each species. The resulting uncertainty in D can broaden thec(s) distribution but typically has little impact on the c(s)maximum.48 The broadening is exaggerated in the c(M)distribution because the molecular weight depends on theratio of s to D.

Divalent Ion Binding Behavior. Panels A and B of Figure4 compare the raw and integrated ITC data, respectively, fromMg2+ titrations of ALG-2des23wt (gray) and D169A (red).Evidently, substitution of alanine for aspartate at position 169

Table 2. Mg2+ Coordination Geometry

Mg2+−Ligand Distances

ligand distance (Å)a

Site 1Asp 36 OD1 2.00(0.02)Asp 38 OD1 2.03(0.02)Ser 40 OG 2.86(0.02)Val 42 O 2.05(0.02)Glu 47 OE1 2.28(0.02)H2O 2.05(0.02)

Site 3Asp 103 OD1 2.09(0.01)Asp 105 OD1 2.04(0.02)Ser 107 OG 2.22(0.03)Met 109 O 2.16(0.01)HOH 5 2.31(0.03)HOH 6 1.90(0.04)

Site 5Asp 169 OD1 2.11(0.02)Asp 171 OD1 2.00(0.03)Asp 173 OD1 2.11(0.03)Trp 175 O 2.06(0.02)HOH 1 1.99(0.03)HOH 2 2.23(0.04)

Angles (ligand 1−Mg2+−ligand 2)

ligand 1 ligand 2 angle (deg)

Site 1Asp 36 OD1 Asp 38 OD1 87.40(0.8)Asp 36 OD1 Ser 40 OG 89.91(0.8)Asp 36 OD1 Val 42 O 92.99(1.7)Asp 36 OD1 Glu 47 OE1 99.54(0.7)Asp 36 OD1 HOH 171.31(1.3)Asp 38 OD1 Ser 40 OG 72.57(1.8)Asp 38 OD1 Val 42 O 147.05(2.1)Asp 38 OD1 Glu 47 OE1 116.05(1.2)Asp 38 OD1 HOH 90.02(1.0)Ser 40 OG Val 42 O 74.48(0.8)Ser 40 OG Glu 47 OE1 167.29(1.3)Ser 40 OG HOH 81.40(1.1)Val 42 O Glu 47 OE1 96.40(1.1)

Angles (ligand 1−Mg2+−ligand 2)

ligand 1 ligand 2 angle (deg)

Site 1Val 42 O HOH 84.68(1.4)Glu 47 OE1 HOH 89.05(1.7)

Site 3Asp 103 OD1 Asp 105 OD1 88.70(0.6)Asp 103 OD1 Ser 107 OG 99.72(0.7)Asp 103 OD1 Met 109 O 94.44(1.5)Asp 103 OD1 HOH 5 175.49(1.8)Asp 103 OD1 HOH 6 92.48(0.8)Asp 105 OD1 Ser 107 OG 90.42(1.0)Asp 105 OD1 Met 109 O 176.27(1.4)Asp 105 OD1 HOH 5 92.80(0.9)Asp 105 OD1 HOH 6 90.33(1.3)Ser 107 OG Met 109 O 87.08(0.5)Ser 107 OG HOH 5 84.52(2.0)Ser 107 OG HOH 6 167.79(1.0)Met 109 O HOH 5 84.22(0.7)Met 109 O HOH 6 91.53(0.5)HOH 5 HOH 6 83.27(1.8)

Site 5Asp 169 OD1 Asp 171 OD1 83.74(1.1)Asp 169 OD1 Asp 173 OD1 87.58(0.8)Asp 169 OD1 Trp 175 O 92.25(0.9)Asp 169 OD1 HOH 1 96.99(1.0)Asp 169 OD1 HOH 2 170.23(1.0)Asp 171 OD1 Asp 173 OD1 85.71(1.2)Asp 171 OD1 Trp 175 O 174.96(1.2)Asp 171 OD1 HOH 1 92.76(1.6)Asp 171 OD1 HOH 2 93.80(0.8)Asp 173 OD1 Trp 175 O 91.09(1.2)Asp 173 OD1 HOH 1 175.00(1.7)Asp 173 OD1 HOH 2 82.80(0.9)Trp 175 O HOH 1 90.75(1.5)Trp 175 O HOH 2 89.64(1.7)HOH 1 HOH 2 92.56(0.6)

aThe numbers displayed in parentheses represent the uncertainties forthe bond distances and angles.

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has eliminated high-affinity Mg2+ binding. The integrated datadisplayed in Figure 4C represent Ca2+ titrations of D169Aconducted in the absence (black) or presence (red) of 0.25 mMMg2+. Whereas inclusion of Mg2+ perturbs the titration of thewild-type protein,35 it has no perceptible impact on the Ca2+

titration of D169A.The Ca2+ binding behavior of D169A was examined by global

least-squares analysis of titration data collected in the absenceand presence of a low-molecular weight chelator (EDTA,EGTA, or NTA). The integrated data from those experiments,displayed in Figure 5, are satisfactorily accommodated by a two-site model. The solid lines depict the best least-squares fit. Theoptimal parameter estimates are listed in Table 3. Presumably,KCa,1 and ΔHCa,1 correspond to EF3, whereas KCa,2 and ΔHCa,2correspond to EF1. For comparison, the corresponding valuesof KCa,1 and KCa,2 measured for the wild-type protein were 8.02× 106 and 6.32 × 105 M−1, respectively.35

Urea Denaturation. When a sample of ALG-2des23wt istitrated at 25 °C with urea in phosphate-buffered KCl (150mM), the protein unfolds with a transition midpoint ofapproximately 5.6 M (Figure 6A, red). Inclusion of 1.0 mMMg2+ shifts the transition midpoint to 6.3 M (green). When asample of the D169A variant is similarly titrated, the transitionmidpoint is at 5.5 M. In contrast to the case for the wild-typeprotein, however, the addition of Mg2+ has no effect on thestability curve. As shown in Figure 6B, the titration curvesobtained in the absence and presence of 1.0 mM Mg2+ areidentical within experimental error.Global Fit of the ITC Data of ALG-2des23wt.

Crystallization of ALG-2des23wt was performed in the presenceof 1.0 mM Mg2+, with the assumption that only the high-affinitysite would be occupied at that concentration. In fact, all threeCa2+-binding sites (EF1, EF3, and EF5) are populated, makingthe Mg2+ binding parameters for the two low-affinity sites amatter of some interest. To obtain estimates for the associationconstants for the low-affinity sites, a series of competition

experiments was performed. Six samples of ALG-2des23wt weretitrated with Ca2+ in the presence of Mg2+. Another five weretitrated with Mg2+ in the presence of Ca2+. Finally, four sampleswere simultaneously titrated with Ca2+ and Mg2+, employingdifferent Mg2+:Ca2+ ratios.The resulting data set was analyzed globally, using the Adair

three-site model described in Materials and Methods. For theleast-squares minimization, the Ca2+ binding parameters werefixed at the previously determined values. The binding constantand enthalpy of the high-affinity Mg2+ site were similarly fixedat their previously determined values. The association constantsand enthalpies for the low-affinity sites were allowed to vary.Likewise, the parameters associated with formation of themixed species (CaMgP, Ca2MgP, and CaMg2P) were allowedto vary.As described in Materials and Methods, the formation

constants for the mixed species were defined as the product ofthe relevant individual association constants, the free ionconcentrations, and a cooperativity index, initially set to 1.0.Similarly, the formation enthalpies were defined as the sum ofthe individual binding enthalpies and a perturbation enthalpy,initially set to 0.

Figure 3. Sedimentation velocity analysis of ALG-2des23wt and ALG-2des23wt/D169A. Samples of the proteins were centrifuged at 30000rpm and 20 °C. The resulting data sets were analyzed with Sedfit toextract (A) the sedimentation coefficient distribution, c(s), and (B) themolecular weight distribution, c(M). The red and green lines representthe data for the wild-type protein and D169A variant, respectively.

Figure 4. D169A eliminates the high-affinity Mg2+-binding site. (A)Direct titration with Mg2+. The red line depicts raw data for thetitration of 63 μM ALG-2des23wt/D169A with Mg2+. The gray line(offset vertically for the sake of clarity) represents the correspondingdata for the wild-type protein. (B) Integrated data for the titrationspresented in panel A. The gray symbols represent the wild-type dataand the red symbols the D169A data. (C) Titration of D169A withCa2+ in the absence (black symbols) and presence (red symbols) of0.25 mM Mg2+.

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The optimal values for the binding parameters are listed inTable 4. Uncertainties were estimated as described in Materialsand Methods (Table 5). The solid lines through the data pointsin Figures 7 and 8 indicate the quality of the fit. Themacroscopic Mg2+ association constants for the two low-affinitybinding events are 43 and 365 M−1, suggesting that occupationof one of the two sites facilitates binding at the second. Thecooperativity index for formation of the CaMgP species is 1.13,an indication that the presence of Mg2+ in EF5 may slightlyfacilitate binding of Ca2+ at the high-affinity site (EF3), and vice

versa. The enthalpy change that accompanies formation ofCaMgP is perceptibly more endothermic (by 0.61 kcal mol−1)than predicted from the enthalpy changes for the independentbinding events. By contrast, the cooperativity index for theCa2MgP species is 0.67, which implies that occupation of EF5by Mg2+ discourages occupation of both high-affinity sites byCa2+. The reverse would also be true, i.e., that occupation of thetwo high-affinity Ca2+ sites would lower, albeit slightly, Mg2+

affinity in EF5. The perturbation enthalpy associated withformation of Ca2MgP is negligible. Finally, the cooperativityindex associated with formation of CaMg2P is nearly unity(0.95), suggesting that the macroscopic cooperativity isminimal. However, formation of the complex is slightly moreexothermic than predicted on the basis of the enthalpy changesfor the independent binding events.

■ DISCUSSIONAlthough the ALG-2 monomer possesses a single high-affinityMg2+-binding site, as assessed by ITC, all three of the EF-handmotifs that have affinity for Ca2+ (EF1, EF3, and EF5) areoccupied by Mg2+ when the protein is crystallized from 0.001

Figure 5. Global analysis of the Ca2+ binding behavior of D169A. Thesolid lines through the data points represent the optimal fit to a two-site Adair model, in which P = [D169A] and C = [Ca2+]: (A) 0.96 mMC vs 60 μM P (red) and 0.96 mM C vs 30 μM P (green), (B) 1.92mM C vs 60 μM P with 100 μM EDTA (cyan) and 1.92 mM C vs 60μM P with 50 μM EDTA (magenta), (C) 1.92 mM C vs 60 μM P with100 μM EGTA (red) and 1.92 mM C vs 60 μM P with 50 μM EGTA(green), and (D) 1.92 mM C vs 60 μM P with 100 μM NTA (cyan)and 1.92 mM C vs 60 μM P with 1.00 mM NTA (magenta).

Table 3. Ca2+ Binding Parameters for ALG-2des23wt/D169A

parameter valuea parameter valuea

KCa,1 5.21(1.19) × 106 ΔHCa,1 −6880(100)KCa,2 2.00(0.35) × 105 ΔHCa,2 −2940(140)

aBinding constants are reported in units of inverse molar andenthalpies in units of calories per mole. Values in parenthesesrepresent the 95% confidence interval.

Figure 6. Mg2+ does not stabilize D169A. (A) ALG-2des23wt (3 μM)was titrated with urea at 25 °C in 0.15 M KCl, 0.01 M KPi, and 0.5mM EGTA (pH 7.4) in the absence (red symbols) or presence (greensymbols) of 1.0 mM Mg2+. (B) D169A (3 μM) was similarly titratedwith urea in the absence (red symbols) or presence (green symbols) of1.0 mM Mg2+.

Table 4. Divalent Ion Binding Parameters for ALG-2des23wt

parameter valuea parameter valuea

KCa,1 8.02(0.89) × 106 ΔHCa.,1 −8430(70)KCa,2 6.32(0.59) × 105 ΔHCa,2 −2210(90)KCa,3 1.09(0.52) × 105 ΔHCa,3 −870(90)KMg,1 5.14(0.11) × 104 ΔHMg,1 1270(50)KMg,2 43(10) ΔHMg,2 1760(190)KMg,3 365(65) ΔHMg,3 −50(10)CICaMg 1.13(0.10) δHCaMg 610(70)CICa2Mg 0.67(0.07) δHCa2Mg 10(10)

CICaMg2 0.95(0.15) δHCaMg2 −240(40)aBinding constants are reported in units of inverse molar andenthalpies in units of calories per mole. Values in parenthesesrepresent the 95% confidence interval.

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Table

5.DataUsedfortheEstim

ationof

Bon

dLength

andBon

dAngle

Uncertainties

beam

line

ALS

4.2.2a

ALS

4.2.2a

ALS

4.2.2a

ALS

4.2.2

ALS

4.2.2

ALS

4.2.2

spacegroup

P212

12P2

1212

P212

12P2

1212

P212

12P2

1212

unitcellparameters(Å)

a=76.5

a=76.5

a=76.5

a=76.4

a=76.3

a=76.6

b=48.5

b=48.5

b=48.5

b=48.6

b=48.4

b=48.5

c=54.3

c=54.3

c=54.3

c=54.3

c=54.3

c=54.3

wavelength(Å)

1.0000

1.0000

1.0000

1.0000

1.0000

1.0000

resolutio

n(Å)

54.35−

1.72

(1.75−

1.72)

54.33−

1.80

(1.84−

1.80)

54.34−

1.75

(1.78−

1.75)

54.31−

1.77

(1.81−

1.77)

54.30−

1.80

(1.84−

1.80)

54.29−

1.80

(1.84−

1.80)

no.o

fobservations

152354

130621

145245

141188

131223

161258

no.o

funique

reflectio

ns22193

19377

21066

20355

19102

19415

Rmerge(I)

0.045(1.014)

0.039(0.754)

0.047(0.938)

0.059(1.029)

0.053(1.095)

0.047(0.921)

Rmeas(I)

0.049(1.103)

0.042(0.839)

0.051(1.014)

0.064(1.121)

0.057(1.187)

0.050(0.999)

Rpim(I)

0.018(0.429)

0.016(0.358)

0.019(0.383)

0.024(0.439)

0.022(0.454)

0.016(0.385)

meanCC1/2

1.000(0.770)

1.000(0.823)

1.000(0.715)

0.999(0.725)

0.999(0.698)

1.000(0.790)

meanI/σ

25.8

(1.6)

29.7(1.9)

25.6(1.8)

18.8(1.5)

20.1(1.5)

25.9

(1.8)

completeness(%

)100.0(100.0)

99.9(100.0)

100.0(100.0)

100.0(100.0)

99.4(98.7)

100.0(100.0)

multip

licity

6.9(6.5)

6.7(5.3)

6.9(6.9)

6.9(6.4)

6.9(6.7)

8.3(6.6)

Rcrystb

0.179−

0.182

0.174−

0.175

0.168−

0.178

0.179−

0.180

0.174−

0.180

0.177−

0.181

Rfreeb,c

0.207−

0.213

0.204−

0.207

0.209−

0.213

0.218−

0.224

0.203−

0.212

0.212−

0.218

rmsd

forbond

lengths(Å)b

0.006−

0.006

0.006−

0.007

0.006−

0.006

0.006−

0.006

0.006−

0.006

0.006−

0.006

rmsd

forbond

angles

(deg)b

0.694−

0.901

0.673−

0.793

0.700−

0.738

0.662−

0.923

0.666−

0.705

0.703−

1.03

coordinate

error(Å)b

,d0.20−0.20

0.18−0.19

0.20−0.21

0.21−0.22

0.21−0.23

0.19−0.20

aDatasetscollected

from

thesamecrystal.bRange

ofvalues

from

10simulated

annealingrefinements.cRandom

5%testset.dMaximum

likelihood-basedcoordinateerrorestim

atefrom

PHENIX

refine.

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M Mg2+. Our previous divalent ion binding study suggestedthat EF5 was the high-affinity Mg2+ site. That hypothesis issupported by the crystal structure. As described below, themajor difference in the tertiary structure provoked by thebinding of Mg2+ is observed at the C-terminal end of theprotein, and of the three sites, the bond lengths and angles inEF5 most closely approach those of ideal Mg2+ coordinationgeometry. To confirm that EF5 was, in fact, the high-affinityMg2+-binding site, D169 was replaced with alanine. Althoughthe resulting variant retains its dimeric quaternary structure, theheat effects that accompany titration with Mg2+ are identical tothose observed upon titration with buffer alone. Consistentwith the apparent absence of Mg2+ binding activity, thepresence of Mg2+ (1.0 mM) does not increase the resistance ofD169A to urea denaturation.The replacement of D169 with alanine also abolishes Ca2+

binding in EF5, so that the ITC data for titration of D169Awith Ca2+ can be satisfactorily treated with a two-site model.This finding conflicts with an earlier report, based on 45Ca2+

flow-dialysis data, that the D169A mutation had a minimalimpact on low-affinity Ca2+ binding by ALG-2.34 Interestingly,the D169A mutation significantly perturbs the Ca2+ affinityassociated with the two remaining sites. The high-affinity sitesof wild-type ALG-2des23wt display Ca2+ association constantsof 8.02 × 106 and 6.32 × 105 M−1. Least-squares analysis ofD169A yielded corresponding estimates of 5.21 × 106 and 2.00× 105 M−1, respectively. Thus, the affinity of the higher-affinity

site, presumably EF3, is reduced by 35%, which corresponds toa ΔΔG value of 0.26 kcal mol−1. The affinity of the other site isreduced by a factor of 3.16, or 0.68 kcal mol−1. This resultsuggests that the high-affinity Ca2+-binding site and, potentially,the target protein-binding surface are sensitive to structuralalterations at the dimer interface. In this context, Subramanianet al.34 noted that the D169A variant exhibited lower affinity forthe ALG-2 target protein, Alix/AIP1.In EF5, the bound Mg2+ ion is coordinated by three

carboxylates (+x, +y, +z), a main-chain carbonyl (−y), and twowater molecules (−x, −z). In ALG-2des23wt, that site displays abinding constant for Mg2+ equal to 51400 M−1 in Hepes-buffered KCl at 25 °C. Intracellular Mg2+ levels can varysubstantially, For example, in cultured dorsal root ganglionneurons, the concentrations in the nucleus and cytoplasm are0.68 ± 0.10 and 0.11 ± 0.05 mM, respectively.49 However, theMg2+ association constant for EF5 is sufficiently high that thesite should be largely occupied independent of intracellularlocation. Even at the low Mg2+ concentration in the neuronalnucleus, EF5 would be 85% occupied.The Mg2+ affinity of EF5 is comparable to that observed for

Ca2+/Mg2+, or “mixed”, sites. In the latter, however, the bound

Figure 7. ALG-2des23wt divalent ion binding. Analysis of the Ca2+ sitesand the high-affinity Mg2+ site. Integrated data are presented, togetherwith solid lines representing the best least-squares fit. Vertical offsetshave been added to permit display in a single figure without overlap.Ca2+ and Mg2+ titrant concentrations were 1.94 and 1.90 mM,respectively, except for those for curve (A)1 (0.94 mM Ca2+) andcurve (B)1 (0.97 mM Mg2+). Protein (P) and chelator concentrationswere as follows. (A) Ca2+ titrations: (1) 32 μM P, (2) 63 μM P, (3) 62μM P with 100 μM NTA, (4) 62 μM P with 1.0 mM NTA, (5) 120μM P, (6) 65 μM P with 50 μM EDTA, (7) 65 μM P with 100 μMEDTA, (8) 65 μM P with 50 μM EGTA, and (9) 62 μM P with 100μM EGTA. (B) Mg2+ titrations: (1) 63 μM P, (2) 109 μM P, (3) 180μM P, (4) 63 μM P with 100 μM EDTA, (5) 112 μM P with 100 μMEDTA, and (6) 189 μM P with 100 μM EDTA.

Figure 8. ALG-2des23wt divalent ion binding. Analysis of the Ca2+ vsMg2+ competition experiments. P = protein; C = Ca2+; M = Mg2+.Ca2+ and Mg2+ titrant concentrations were 1.94 and 1.90 mM,respectively. (A) Titrations with Ca2+ in the presence of Mg2+: (1) 115μM P and 120 μM M, (2) 116 μM P and 235 μM M, (3) 116 μM Pand 0.49 mM M, (4) 120 μM P and 0.99 mM M, (5) 115 μM P and2.92 mM M, and (6) 120 μM P and 9.90 mM M. (B) Titrations withMg2+ in the presence of Ca2+: (1) 114 μM P and 63 μM C, (2) 116μM P and 120 μM C, (3) 116 μM P and 180 μM C, (4) 124 μM Pand 250 μM C, and (5) 122 μM P and 306 μM C. (C) Simultaneoustitrations with Ca2+ and Mg2+: (1) 0.97 mM C and 0.95 mM M, (2)0.97 mM C and 1.90 mM M, (3) 0.97 mM C and 3.80 mMM, and (4)0.97 mM C and 7.60 mM M. The protein concentration was 60 μM ineach experiment.

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ion is ligated by four carboxylates positioned (most commonly)at +x, +y, +z, and −z or (in the parvalbumin CD site) at +x, +y,−x, and −z. Ca2+/Mg2+ sites prefer Ca2+ over Mg2+ by a factorof 103−104.1 By contrast, the EF5 site in ALG-2des23wt favorsCa2+ by a ratio of just 2.2. In ALG-2des23ΔGF122, where Mg2+

should be similarly coordinated, the ratio is only 0.53. It wouldbe interesting to examine the behavior of the EF5 coordinationsphere in other EF-hand protein systems, given that it appearsto offer a blueprint for an EF-hand motif having high affinityand selectivity for Mg2+. It should be noted that the EF5 siteharbors a one-residue insertion near the C-terminal end of thebinding loop and that, in the absence of the insertion, thesequence of EF5 resembles that of a typical Ca2+/Mg2+ site.In this context, we can ask whether other penta-EF-hand

family members might possess a high-affinity Mg2+ site. PEFproteins are assigned to either group I (ALG-2 and peflin) orgroup II (sorcin, grancalcin, and calpain) based on thesequence within EF1. Whereas group I proteins display acanonical EF-hand motif, the binding loop in group II harborsan Asp → Ala substitution at +x, as well as a one-residuedeletion. Figure 9 displays aligned sequence data for the EF5

motifs in a subset of PEF proteins. On the basis of comparisonwith ALG-2, we would predict that the following proteins donot possess a high-affinity Mg2+ site: peflin, sorcin, grancalcin,and the small subunit of calpain. However, the sequences of atleast two of the calpain large subunit isoforms, CAPN1 andCAPN8, contain the triad of aspartyl residues at +x, +y, and +zbelieved to be required for high-affinity Mg2+ binding.The high-affinity Ca2+ sites have low affinity for Mg2+. The

macroscopic stepwise binding constants for the two low-affinityMg2+ binding events, KMg,2 and KMg,3, obtained from least-squares fitting are 43 and 365 M−1, respectively. The largervalue of Kmg,3 suggests that there is some cooperativityassociated with the second and third Mg2+ binding events;i.e., occupation of one of the high-affinity Ca2+ sites by Mg2+

facilitates occupation of the other. However, at a free Mg2+

concentration of 1.0 mM, the predicted fractional populationsof MgP, MgP2, and MgP3 would be 0.94, 0.04, and 0.02,respectively. Thus, the interaction of Mg2+ with EF1 and EF3 isunlikely to have any physiological relevance.

Given their low affinity for Mg2+, the occupancy of EF1 andEF3 in the crystal structure was unexpected. The explanationmay reside in the polyanionic character of ALG-2. The pH ofthe 0.10 M Tris solution employed for crystallization was 7.4 at25 °C. At 4 °C, the temperature at which the crystallization wasperformed, the pH would be approximately 8.0. The predictedcharge on ALG-2des23wt at pH 8.0 is −4.4. Occupation of allthree EF-hands may be required to sufficiently reduce the levelof intermolecular electrostatic repulsion in the 2-propanol/water solvent so that nucleation and subsequent crystal growthcan occur. Additionally, the monomer concentration of ALG-2in the Mg2+-bound crystals is approximately 0.032 M. As aresult, the anion concentration in the crystal would exceed thatof the bulk crystallization liquor by roughly 4.4 × 0.032 M, or0.13 M. Thus, it is likely that the Mg2+ concentration in thecrystal environment substantially exceeds that in the bulkcrystallization liquor, which would also tend to promoteoccupation of EF1 and EF3.Consistent with its low affinity for Mg2+, the coordination

environment observed in EF1 departs significantly from idealMg2+ coordination geometry. FindGeo classifies the site astrigonal bipyramidal, rather than octahedral, presumablybecause of the large bond distance (2.86 Å) between Oγ ofS40 and the bound metal ion. The basis for the low affinity ofEF3 is less apparent. FindGeo recognizes the Mg2+

coordination as octahedral, and the bond distance rmsd,relative to ideal Mg2+ coordination, at 0.22 Å, is only slightlylarger than that calculated for EF5 (0.20 Å). The ligand arraysin both EF5 and EF3 include two water molecules. In EF3,however, a serine hydroxyl occupies the +z position, rather thanan aspartyl carboxylate, as in EF5. The additional anionic ligandin EF5 may be responsible for its much higher avidity for Mg2+.The tertiary structure of the Mg2+-bound ALG-2des23wt

monomer closely resembles that of the Ca2+-free protein(PDB entry 2ZND), in which EF1, EF3, and EF5 are occupiedby Na+ ions. As shown in Figure 10A, the Cα traces are virtuallycoincident, with the only major departure occurring at the C-terminus. The overall rmsd for the two structures is 0.64 Å.The dimer interfaces bury comparable surface areas in the

two structures, 1460 Å2 for the Ca2+-free protein and 1410 Å2

for the Mg2+-bound form. However, the orientation of themonomers differs perceptibly in the two structures. As shownin Figure 10C, the separation between the subunits issomewhat more pronounced in the Mg2+-bound dimer. As aresult, the Mg2+-bound protein has a slightly larger radius ofgyration, 24.1 Å, versus 23.1 Å for the Ca2+-free form. The shiftin the relative monomer position is accompanied by modifiedside-chain packing at the interface, also illustrated in Figure10C. Note that the V191 residues from opposing subunits areadjacent when Mg2+ occupies EF5. However, when EF5 isoccupied by Na+, they rotate apart.In both structures, V191 from one subunit contacts K137 in

the opposing subunit. However, in the Mg2+-bound protein, theorientation of V191 allows the C-terminal carboxylate to form asalt bridge with the ε-ammonium group of K137. Figure 10Bdepicts this interaction in cartoon form. Additionally, theorientation in the Mg2+-bound form allows the backbonecarbonyl of F188 to hydrogen bond with the ε-ammoniumgroup of K137. Because of its location near the C-terminal endof the helix, the F188 carbonyl cannot form an intrahelicalhydrogen bond. Thus, the interaction with K137 reflectsformation of an additional hydrogen bond, rather than merelyexchange of one for another.

Figure 9. EF5 sequences in select human penta-EF-hand proteins.ALG-2 (GenBank entry AAC27697.1) and peflin (NCBI ref SeqNP_036524.1) belong to group I. Sorcin (GenBank entryAAA92155.1), grancalcin (NCBI ref Seq NP_036330.1), and calpainbelong to group II. CAPN4 (NCBI ref Seq NP_001003962.1)represents the small subunit of human calpain. CAPN1 (GenBankentry AAH75862.1), CAPN2 (GenBank entry AAH21303.1), CAPN8(GenBank entry AAI57894.1), and CAPN11 (GenBank entryAAH33733.1) correspond to four of the 12 human isoforms of thelarge subunit of calpain.

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Apparently, the binding of Ca2+ in EF5 does not similarlyinfluence the orientation of the C-terminal helix. There are twoCa2+-bound ALG-2 structures in the PDB in which both EF5sites are fully occupied (1HQV50 and 2ZN936). Although theCa2+ coordination in EF5 resembles that of Mg2+, the C-terminiare not visible in either structure, suggesting that they samplemultiple conformations. The low Ca2+ affinity of EF5 precludesappreciable occupation at intracellular levels of the ion.However, the X-ray diffraction data imply that, even if theEF5 sites were completely filled, the impact of Ca2+ binding onthe C-terminal helix would not be equivalent to that attendantto Mg2+ binding.It is at present unknown whether the Mg2+-promoted

interactions between F188/V191 and K137 from opposingmonomers are functionally significant. The dimeric structure ofALG-2 is required for its putative function as a molecularadaptor. Although ALG-2des23wt and the D169A variant areboth dimeric in the absence of divalent cations at the relativelyhigh concentration (22 μM) employed in our sedimentationanalysis, ALG-2 reportedly dimerizes more weakly than otherpenta-EF hand proteins, such as sorcin and grancalcin.13 Lo etal.33 detected monomeric and dimeric species at micromolarconcentrations in vitro, and Subramanian et al.34 suggested thatthe dissociation constant for the Ca2+-free ALG-2 dimer was inthe low micromolar range. Thus, at physiological concen-trations of the protein, the self-association of apo-ALG-2 maybe incomplete. It is likely that the Mg2+-promoted interactionsbetween opposing monomers would stabilize the ALG-2 dimer.

■ CONCLUDING REMARKSEF5, the high-affinity Mg2+ site in ALG-2, harbors a single-residue insertion. As noted above, the insertion distinguishesEF5 from a consensus Ca2+/Mg2+-binding loop. The additionalresidue prevents E180 from functioning as the canonical −zligand and creates a vacancy that is filled by a water molecule.This change in coordination environment seriously compro-mises Ca2+ affinity but leaves the Mg2+ affinity intact.Occupation of EF5 by Mg2+, but evidently not Ca2+, producesa reorientation of the C-terminal helix that is predicted tostabilize the ALG-2 dimer.The selective pressure for the insertion in EF5 is uncertain.

In its absence, glutamate could serve as the −z ligand,presumably yielding an EF-hand motif with very high affinityfor both Ca2+ (≥107 M−1) and Mg2+ (≥104 M−1). However,this conventional Ca2+/Mg2+ site might not similarly promoteALG-2 dimer formation. For example, insertion of an additionalcarboxylate into the metal ion coordination spheres of thejuxtaposed EF5 sites could electrostatically destabilize thedimer. Alternatively, glutamyl coordination at −z might preventthe C-terminal helix from adopting the orientation required forthe interactions between F188/V191 and K137 from theopposing monomer. In this context, it is noteworthy that the −z glutamate is a monodentate ligand to Mg2+ and a bidentateligand to Ca2+. Thus, the C-terminus might be optimallypositioned to promote dimerization when EF5 is occupied byMg2+ at resting-state levels of Ca2+. However, the exchange ofCa2+ for Mg2+ that would occur when the intracellular Ca2+

level is elevated could perturb the alignment of the C-terminalhelix, destabilizing the dimer and compromising ALG-2 adaptorfunction. Clearly, the rationale for incorporating an unusual EF-hand motif with high affinity and selectivity for Mg2+ at theALG-2 dimer interface is an issue that deserves furtherattention.

■ ASSOCIATED CONTENTAccession CodesCoordinates and structure factors have been deposited in theProtein Data Bank as entry 5JJG.

■ AUTHOR INFORMATIONCorresponding Author*Department of Biochemistry, 117 Schweitzer Hall, Universityof Missouri, Columbia, MO 65211. Telephone: 573-882-7485.Fax: 573-884-4812. E-mail: henzlm@missouri.edu.FundingThis work was supported by the Department of Biochemistry atthe University of Missouri.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Dr. Jay Nix for help with X-ray data collection. Partof this research was performed at the Advanced Light Source,which is supported by the Director, Office of Science, Office ofBasic Energy Sciences, of the U.S. Department of Energy underContract DE-AC02-05CH11231.

■ ABBREVIATIONSABM, ALG-2-binding motif; EDTA, ethylenediaminetetraaceticacid; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1-piper-

Figure 10. Comparison of the apo (Na+-bound) and Mg2+-boundALG-2 structures. (A) Superposition of the Cα traces of apo (red) andMg2+-bound (silver) ALG-2des23wt. (B) Ionic and hydrogen-bondinginteractions between V191 and K137 from opposing subunits at thedimer interface. (C) Surface representations of the Mg2+-bound (left)and apo (right) ALG-2des23wt dimers.

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azineethanesulfonic acid; IPTG, isopropyl β-D-thiogalactopyr-anoside; ITC, isothermal titration calorimetry; LB, Luria-Bertani; NTA, nitrilotriacetic acid; PEF, penta-EF-hand; PRR,proline-rich region; rmsd, root-mean-square deviation; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electro-phoresis.

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