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IDENTIFICATION OF LIGAND BINDING SITE OF PHYTOSULFOKINERECEPTOR BY ON-COLUMN PHOTOAFFINITY LABELING*
Hidefumi Shinohara, Mari Ogawa, Youji Sakagami and Yoshikatsu MatsubayashiFrom the Graduate School of Bio-Agricultural Sciences, Nagoya University
Chikusa, Nagoya 464-8601, JAPANRunning Title: Ligand Binding Site of Phytosulfokine Receptor
Address correspondence to: Y. Matsubayashi. Graduate School of Bio-Agricultural Sciences, NagoyaUniversity, Chikusa, Nagoya 464-8601, JAPAN, Tel.: +81-52-789-4117; Fax.: +81-52-789-4118; E-mail: [email protected] (Y. Matsubayashi)
Phytosulfokine (PSK), an endogenous 5-amino-acid secreted peptide in plants, affectscellular potential for growth via binding toPSKR1, a member of the leucine-rich repeatreceptor kinase (LRR-RK) family. PSKinteracts with PSKR1 in a highly specificmanner with a nanomolar dissociationconstant. However, it is not known whichresidues in the PSKR1 extracellular domainconstitute the ligand-binding pocket. Here, weidentified the PSK-binding domain of carrotPSKR1 (DcPSKR1) by photoaffinity labeling.We cross-linked the photoactivatable PSKanalog [125I]N -[(4-azidosalicyl)Lys5]PSK withDcPSKR1 using UV irradiation, and mappedthe cross-linked region using chemical andenzymatic fragmentation. We also establisheda novel “on-column photoaffinity labeling”methodology that allows repeatedincorporation of the photoaffinity label toincrease the efficiency of the photoaffinitycross-linking reactions. We purified a labeledDcPSKR1 tryptic fragment using anti-PSKantibodies, and identified a peptide fragmentthat corresponds to the 15-amino-acid Glu503-Lys517 region of DcPSKR1 by MALDI-TOFMS. Deletion of Glu503-Lys517 completelyabolishes the ligand-binding activity ofDcPSKR1. This region is in the island domainflanked by extracellular LRRs, indicatingthat this domain forms a ligand-bindingpocket that directly interacts with PSK.
Cell-to-cell communication is essential forgrowth and development of multicellularorganisms throughout their life. In plants,hormones including small lipophilic compoundsand secreted peptides comprise a large group ofsignaling molecules that are central tointercellular communication. They elicitbiological activity by binding to cell surfacereceptors that have kinase activity, or by directlyinteracting with intracellular proteins.
Phytosulfokine (PSK) is a 5-amino-acidsecreted peptide that has been identified in themedium of plant cell cultures, based on theresults of assays of growth-promoting activity ofcultured cells (1). Addition of chemically
synthesized PSK to culture medium, even atnanomolar concentrations, significantlypromotes the proliferation of callus andsuspension cells. PSK also promotes trachearyelement differentiation (2), somaticembryogenesis (3,4), adventitious bud formation(5), adventitious root formation (6) and pollengermination in vitro (7). PSK is produced from≈80-amino-acid precursor peptides via post-translational sulfation of tyrosine residues andproteolytic processing (8). Genes encoding PSKprecursors are redundantly distributed in thegenome, and are expressed in cultured cells andin a variety of tissues including leaves, stems,flowers and roots (9,10).
PSK binds the membrane-localized PSKreceptor PSKR1, which is a leucine-rich repeatreceptor kinase (LRR-RK) that has been purifiedfrom solubilized carrot microsomes by ligand-based affinity chromatography (hereafterreferred to as DcPSKR1) (11). The extracellulardomain of DcPSKR1 contains 21 tandem copiesof LRR interrupted by a 36-amino-acid islanddomain rich in hydrophilic and charged aminoacid residues. Disruption or overexpression ofthe Arabidopsis ortholog of PSKR1 (AtPSKR1)significantly alters cellular longevity andpotential for growth without interfering withprimary morphogenesis of plants (10). PSKappears to activate the basic potential for cellulargrowth rather than directly determining cell fate,and thereby exerts a pleiotropic effect onindividual cells in response to environmentalhormonal conditions.
Ligand binding generally causes a receptorprotein to undergo a conformational change thatdirectly activates the receptor so that it caninteract with another cellular molecule and/orexert intrinsic enzyme activities such as kinaseactivity. PSK interacts with DcPSKR1 in ahighly specific manner, with a high-affinitydissociation constant of Kd = 4.2 nM (11).However, it is not known which amino acids inthe DcPSKR1 extracellular domain constitutethe ligand-binding pocket.
Photoaffinity labeling is one of the mostuseful methods for analyzing ligand-receptorinteractions. Identification of the labeled amino
http://www.jbc.org/cgi/doi/10.1074/jbc.M604558200The latest version is at JBC Papers in Press. Published on November 8, 2006 as Manuscript M604558200
Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
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acid residues can yield valuable informationabout the ligand-binding domain of the receptor.However, it is often quite difficult to identify thecross-linked residues by MS analysis, due to thelow efficiency of cross-linking reactions. Thesedifficulties are further compounded by lowconcentrations of binding proteins, especially inthe case of transmembrane receptors andchannels that can not be functionallyoverexpressed in bacteria. In fact, most knowncross-linked regions of membrane-localizedproteins have been identified by analyzingchemical and enzymatic fragmentation patternsof labeled proteins using radioactivephotoaffinity ligands, rather than direct MSanalysis of purified peptide fragmentscrosslinked with photoaffinity ligands (12-14).
In the present paper, we report identificationof the PSK-binding site of DcPSKR1 by SDS-PAGE mapping of the cross-linked fragmentsgenerated by chemical and enzymaticfragmentation of the photoaffinity-labeledligand-receptor complex, and by direct analysisof the purified cross-linked fragments byMALDI-TOF MS. We also report the usefulnessof a novel solid-phase photoaffinity labelingtechnology, “on-column photoaffinity labeling”,which allows repeated incorporation of aphotoaffinity label to increase the efficiency ofthe photoaffinity cross-linking reactions.
[Experimental Procedures]
Vector construction and transformation ofTobacco BY-2 cells––For construction ofplasmids pBI121-DcPSKR1-His6 and pBI121-DcPSKR1-∆KD-His6, PCR was performed usingprimers PSKR-5f (5’-GCtctagaATTTGCCTTGTTTTGTTGAGC-3’)and PSKR-3r (5’-CTAGTGGTGGTGGTGGTGGTGACTACTGACATCAATGTTTTCGAGCC-3’) for DcPSKR1-His6, using primers PSKR-5f and PSKR-3t (5’-CTAGTGGTGGTGGTGGTGGTGACTGCTAGTGGATTTCAAAATGTC-3’) for DcPSKR1-∆KD-His6 (His6-coding regions are underlined;restriction sites are in lowercase), and usingDcPSKR1 cDNA as a template. The amplifiedfragments were digested with XbaI and insertedinto the binary vector pBI121, which had beendigested with XbaI and SacI (blunted). Forconstruction of plasmid pBI121-DcPSKR1-∆ID[Glu503-Lys517], the PCR-ligation-PCRmethod was used (15). Two independent PCRswere performed: 1) using primers PSKR-5f andDcR1ID-3e (5’-GGAGACAAGGCTCTGTAAACTGG-3’); and2) using primers DcR1ID-5e (5’-
AAAAACACAAATGCCGGAGG-3’) andPSKR-3p (5’-AATGACAGGACAATGCTAACTACTGACATC-3’). The two PCR products were thenphosphorylated and blunt ligated. A secondseries of PCR performed to amplify the ligatedDcPSKR1-∆ID[Glu503-Lys517] using primersPSKR-5f and PSKR-3p. Finally, the product waspurified, digested with XbaI and inserted into thebinary vector pBI121, which had been digestedwith XbaI and SacI (blunted). For constructionof pBI121-DcPSKR1-∆ID[Lys518-Ile538], twoindependent PCRs were performed: 1) usingprimers PSKR-5f and DcR1ID-3d (5’-TTTTTTCTTGAAAAATGGAAAATCTGG-3’); and 2) using primers DcR1ID-5d (5’-ATAGACCTTAGTTATAATTCCCTCAATGG-3’) and PSKR-3p. The PCR products werephosphorylated, blunt ligated, and subjected to asecond PCR using primers PSKR-5f and PSKR-3p. The products was purified, digested withXbaI, and inserted into the binary vector pBI121.The resultant recombinant plasmids were used totransform Agrobacterium tumefaciens strainC58C1 (pMP90) using electropolation. TobaccoBY-2 cells were co-cultivated withAgrobacterium for 2 days, and the resultanttransformed BY-2 cells were selected onmodified MS agar medium containing 200 mg/lkanamycin and 500 mg/l carbenicillin for 3 to 4weeks until transformed cells were formed (16).Selected cell lines were transferred into MSliquid medium containing 100 mg/l kanamycinto initiate suspension culture, and were used forsubsequent analysis. Transgenic BY-2 cells weremaintained in both liquid and solid culture bysubculturing once a week.
Ligand binding assay––The ligand binding assayof plant microsomal fractions and affinity-purified DcPSKR1 was performed using[3H]PSK and following a protocol describedpreviously (11,17).
Affinity purification of DcPSKR1- KD-His6––Transformed BY-2 microsomalmembranes (1,800 mg protein prepared from 6-day-old culture) were solubilized in 320 ml ofbuffer containing 20 mM HEPES-KOH (pH 7.5),50 mM KCl, and 1.0% Triton X-100.Solubilized materials were centrifuged at100,000 g for 30 min at 4°C, and thesupernatants were applied to a [Lys5]PSK-Sepharose column (5.0-ml column) at a flowrate of 0.5 ml/min using the AKTA primechromatography system (Amersham PharmaciaBiotech) (11). After washing with 50 ml ofbuffer containing 20 mM HEPES-KOH (pH 7.5),
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50 mM KCl, and 0.1% Triton X-100 (washbuffer), the column was eluted with 15 ml ofbuffer containing 20 mM HEPES-KOH (pH 7.5),500 mM KCl, and 0.1% Triton X-100 (elutionbuffer). The eluates were added to a 1.0-mlMacro-Prep Ceramic Hydroxyapatite Type Icolumn (Bio-Rad laboratories) at a flow rate of0.5 ml/min at 4°C. The column was washed with20 ml of wash buffer and eluted with an 18-mlgradient of KH2PO4 (0 to 400 mM) in washbuffer. Aliquots of active fractions (1.0 ml), asdetermined by [3H]PSK binding assay, wereconcentrated by ultrafiltration (Ultrafree-MCMWCO 30,000, Millipore) and analyzed bySDS-PAGE using 7.5% gels for confirmation ofpurity. Protein bands in SDS-PAGE gels werevisualized using the fluorescent dye SYPRORed (Molecular Probe). The immunoblot of theactive fraction (20 µl) was probed with anti-DcPSKR1 antibodies raised against theDcPSKR1 100-amino-acid N-terminal region(11), and was visualized using ECL Advance(Amersham Pharmacia Biotech) according to themanufacturer’s protocol.
PNGase F treatment of purified DcPSKR1-KD-His6––Affinity-purified DcPSKR1-∆KD-
His6 (10 µl) was added to PNGase F buffercontaining 100 mM Tris-HCl buffer (pH 8.6)and 0.1 % SDS, and was incubated at 95°C for 3min. The denatured sample was then incubatedwith 2 milliunits of peptide N-glycosidase F(PNGase F, TakaraBio, Shiga, Japan) in thepresence of Nonidet P-40 (1.0% final solution)at 37°C for 16 h at a total volume of 25 µl. Afterdeglycosylation, samples were mixed with SDS-PAGE sample buffer, and were then analyzed bySDS-PAGE and immunoblotting, as describedabove.
Preparation of photoactivatable analog of PSK––Preparation of the photoactivatable PSKanalog Nε-[(4-azidosalicyl)Lys5]PSK (ASA-PSK) and radioactive [125I]ASA-PSK wasperformed as previously described (18). Thespecific radioactivity of [125I]ASA-PSK wasestimated to be 210 Ci/mmol.
In-solution photoaffinity labeling––Aliquots ofpurified DcPSKR1-∆KD-His6 (10 µl) wereincubated with 1 µM [125I]ASA-PSK for 10 minat 4°C. Then, the samples were irradiated with aUV lamp (model ENF-260C/J [365 nm],Spectronics Co. Ltd, NY) for 10 min on ice at adistance of <1 cm. SDS-PAGE sample bufferwas added to each of the samples, which werethen heated at 95°C for 5 min. Samples wereloaded onto NuPage 12% Bis-Tris Gel
(Invitrogen) and separated according to themanufacturer’s protocol. The dried gels wereexposed to a bio-imaging plate (Fujifilm, Tokyo,Japan) for 16 h at room temperature, and wereanalyzed using a bio-imaging analyzer (BAS2000, Fujifilm, Tokyo, Japan).
Chemical cleavage and protease digestion––[125I]ASA-PSK-labeled DcPSKR1-∆KD-His6,with or without PNGase F treatment, was usedfor chemical cleavage and protease digestion.Cyanogen bromide (CNBr) cleavage of cross-linked proteins (100 µl) was performed in 75%formic acid containing 0.5 mg/ml CNBr under anitrogen atmosphere. After this solution wasincubated for 16 h at room temperature, it wasdiluted 10-fold with distilled water andlyophilized. For trypsin digestion, 10 µl oflabeled DcPSKR1-∆KD-His6 was treated with100 pmol of TPCK-treated trypsin (Sigma) inbuffer containing 20 mM Tris-HCl (pH 7.6), 10mM CaCl2 and 10% CH3CN (v/v) at 37°C for 16h at a total volume of 20 µl. For Asp-N digestion,10 µl of labeled DcPSKR1-∆KD-His6 wastreated with 100 pmol of Endoproteinase Asp-N(TakaraBio, Shiga, Japan) in 50 mM sodiumphosphate buffer (pH 8.0) at 37°C for 16 h at atotal volume of 20 µl. All of these samples wereanalyzed by SDS-PAGE using the NuPage 12%Bis-Tris Gel and autoradiography.
On-column photoaffinity labeling and trypsindigestion––Affinity-purified DcPSKR1-∆KD-His6 (9 ml) was immobilized on Ni2+-loadedHiTrap chelating HP Sepharose (bed volume,200 µl, Amersham Pharmacia Biotech) byrecycling 3 times. After the column was washedwith 1 ml of the above-described wash buffer, 1ml of 10 nM [ 125I]ASA-PSK or 1 µM cold ASA-PSK (dissolved in the wash buffer) was loadedonto the column, which was then left to stand for5 min for ligand binding. After the column waswashed with 600 µl of the wash buffer toremove unbound ligand, the bound ligand wascross-linked to DcPSKR1-∆KD-His6 by directlyirradiating the column with UV (365 nm) for 10min. Un-cross-linked ligand was removed bywashing the column with 1 ml of the washbuffer, and the labeling cycle was then repeatedup to 3 times for [125I]ASA-PSK and 10 timesfor ASA-PSK. For trypsin digestion, labeledDcPSKR1-∆KD-His6 on Sepharose wassuspended in 200 µl of digestion buffercontaining 20 mM Tris-HCl (pH 7.6), 10 mMCaCl2 and 10% CH3CN in a total volume, andwas then digested by 100 pmol of TPCK-treatedtrypsin (Sigma) at 37°C for 16 h. Sepharose wasthen removed by filtration, and liberated
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peptides were used for the following analysis.
Gel filtration of the labeled trypticpeptides––Gel filtration analysis was performedusing Sephadex G-25 Superfine (1.5 cm ID x 35cm, Amersham Pharmacia Biotech) at a flowrate of 0.2 ml/min. Column equilibration andchromatography were performed using buffercontaining 20 mM HEPES-KOH (pH 7.0) and150 mM NaCl. On-column tryptic digests of[125I]ASA-PSK-labeled DcPSKR1-∆KD-His6
(200,000 cpm eq.) were applied to the column,and 1.0-ml fractions were analyzed forradioactivity using an auto well gamma system(ALOKA). Blue dextran (Amersham PharmaciaBiotech) and cyanocobaramin were used formolecular mass calibration.
Immunoaffinity purification of the labeled trypticpeptides––For the purification of the anti-PSKantibodies, anti-PSK antiserum (5 ml) (19) wasloaded onto a [Lys5]PSK-Sepharose column(1.0-ml column). After the antiserum wasrecycled 3 times, the column was washed withPBS, and the bound antibodies were then elutedwith 0.1% formic acid (pH 2.6) and immediatelyneutralized with NaHCO3. Purified antibodieswere coupled with HiTrap NHS-activated HPSepharose (Amersham Pharmacia Biotech) for16 h at 4°C. The immobilized anti-PSKantibodies were stored in PBS until use. On-column tryptic digests of ASA-PSK-labeledDcPSKR1-∆KD-His6 were incubated at 95°C for10 min to inactivate trypsin, and were thenloaded onto the immuno-affinity column (20-µlcolumn). After the column was washed with 200µl of the trypsin digestion buffer and 200 µl ofdistilled water, bound fragments were elutedwith 1% trifluoroacetic acid (v/v). The eluatewas used for MALDI-TOF MS analysis.
MALDI-TOF MS analysis––The eluate from theimmuno-affinity column was concentrated byevaporation, desalted using Zip Tip C18TM
pipette tips (Millipore), and mass-profiled with a4700 proteomics analyzer (Applied Biosystems)using α-cyano-4-hydroxycinnamic acid (CHCA)as the matrix. A pulsed nitrogen laser (337 nm)was used to induce desorption/ionization, andmass spectra were obtained using reflector mode.Each representative mass spectrum shown in thepresent figures was the smoothed average of100,000 laser shots.
[Results]
Overexpression of DcPSKR1 in Tobacco BY-2cells––To obtain the relatively large amount of
DcPSKR1 protein required for the directidentification of the PSK-binding domain byphotoaffinity labeling, we first overexpressedDcPSKR1 in BY-2 suspension cells, which growrapidly and can be harvested weekly. ForDcPSKR1 expression, BY-2 cells weretransformed using two constructs:35S::DcPSKR1-His6, as a positive control forfunctional expression of full-length DcPSKR1 inheterologous cells; and the 35S::DcPSKR1-∆KD-His6 construct, in which the coding regionfor the kinase domain of DcPSKR1 had beenremoved (Fig. 1A). The latter construct was usedto test whether the extracellular domain ofDcPSKR1 is sufficient for interaction with PSK.The His6 tag was introduced to immobilize theseproteins on the Ni-column to perform on-columnphotoaffinity labeling (described below).
Northern blot and immunoblot analysis of themembrane fractions of each transformed BY-2cell clone revealed that both DcPSKR1-His6 andDcPSKR1-∆KD-His6 proteins were successfullyoverexpressed in BY-2 cells (Fig. 1B). Bothproteins migrated, with an apparent molecularsize of 150 kDa and 120 kDa, respectively. Thetheoretical molecular masses of these proteins,without the signal sequence, are 110.2 kDa and78.8 kDa, respectively, indicating that bothproteins are post-translationally modified byaddition of an approximately 40-kDa moiety,most likely by glycosylation.
The ligand-binding assay using [3H]PSKconfirmed a significant increase in PSK-bindingactivity in the membrane fractions derived fromboth transformants, compared with fractionsderived from untransformed BY-2 cells (Fig.1C). These results indicate that DcPSKR1 isfunctionally expressed in BY-2 cells, and thatthe intracellular kinase domain of DcPSKR1 isnot essential for PSK binding. Scatchardanalysis of the binding of [3H]PSK tomembranes expressing DcPSKR1-∆KD-His6
showed that the dissociation constant (Kd) ofDcPSKR1-∆KD-His6 is 2.1 ± 0.3 nM, which iscomparable with that of wild-type DcPSKR1(4.2 ± 0.4 nM) (11) (Fig. 1D). Because thistruncated DcPSKR1 lacks the kinase domain,and therefore theoretically yields fewer peptidefragments after chemical and/or enzymaticdigestion, we used DcPSKR1-∆KD-His6 in thefollowing experiments.
Purification and photoaffinity labeling ofDcPSKR1- KD-His6––We purified DcPSKR1-∆KD-His6 protein from solubilized microsomalfractions derived from transgenic BY-2 cells,using [Lys5]PSK-Sepharose and hydroxyapatitecolumn chromatography. The relative expression
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level of DcPSKR1-∆KD-His6 protein wasestimated to be 190 fmol/mg microsomalproteins, based on Scatchard analysis. SDS-PAGE analysis of the purified fractionsconfirmed that DcPSKR1-∆KD-His6 protein hadbeen recovered at relatively high purity (Fig. 1Eleft panel). After the PNGase F treatment, theapparent molecular mass of DcPSKR1-∆KD-His6 changed from 120 kDa to approximately110 kDa, indicating that at least 10 kDa of thepresumed 40-kDa posttranslationally addedmoiety is composed of N-linked glycans (Fig.1E right panel). It has been reported that α1,3-fucosylated glycans, which are often found in N-linked glycans in plants, are resistant to PNGaseF treatment (20). Incubation of the purifiedDcPSKR1-∆KD-His6 with 10 nM [125I]ASA-PSK (photoaffinity ligand) (Fig. 2A), followedby cross-linking by UV irradiation, resulted inspecific labeling of the 120-kDa band thatcorresponds to DcPSKR1-∆KD-His6. Theincorporation of [125I]ASA-PSK into the 120-kDa band was completely abolished by thepresence of excess unlabeled PSK, indicatingspecific cross-linking between [125I]ASA-PSKand DcPSKR1-∆KD-His6 (Fig. 2B).
Chemical fragmentation and enzymaticdigestion of [125I]ASA-PSK-labeled DcPSKR1-
KD-His6––Extracellular domain of DcPSKR1contains 21 LRRs with a 36-amino-acid islanddomain between the 17th and 18th LRR. Toroughly map the crosslinked region withinDcPSKR1-∆KD-His6, we chemically andenzymatically fragmented the [125I]ASA-PSK-labeled protein and estimated the apparentmolecular size of the radioactive fragments.
Treatment of labeled DcPSKR1-∆KD-His6
with CNBr, which cleaves polypeptides at theN-terminal of Met residues (21), and subsequentanalysis by SDS-PAGE revealed a labeled 45-kDa polypeptide (Fig. 2B). After the PNGase Ftreatment, the apparent molecular mass of thisband changed from 45 kDa to approximately 35kDa, indicating that a 10-kDa N-linked glycanmoiety is attached to this fragment. After trypticdigestion of this band, MALDI-TOF MSanalysis showed a signal at m/z 1752.8, whichcorresponds to Glu503-Lys517, indicating that thisfragment contains the island domain (data notshown). Because the presence of the Met-Thrsequence causes conversion of Met tohomoserine without cleavage, we speculated thatthe 45-kDa polypeptide (35 kDa after PNGasetreatment) corresponds to the Pro295-Met537
fragment (Fig. 2C). We confirmed that both ofthe two Met-Thr sequences within DcPSKR1-∆KD-His6 are resistant to CNBr treatment, using
bacterially expressed recombinant DcPSKR1-∆KD-His6 (data not shown). The theoreticalmolecular size of this fragment is 26.6 kDa,indicating that it contains severalposttranslationally added moieties which areresistant to PNGase F treatment.
Digestion of labeled DcPSKR1-∆KD-His6 byendoproteinase Asp-N, which cleavespolypeptides at the N-terminal of Asp residues(5 sites within Pro295-Met537 fragment) yieldedfast-migrating labeled peptides with anapproximate size of 5 kDa, with or withoutPNGase F treatment (Fig. 2B), suggesting thatthe crosslinking site is in the C-terminal regionof the CNBr fragment (Fig. 2C). In contrast, thelabeled fragment obtained by trypsin digestion(18 sites within Pro295-Met537 fragment) was nolonger detectable on the gel, indicating that themolecular size of the labeled fragment is <3.5kDa (Fig. 2B). These results indicate that thecrosslinking site is confined to one trypticfragment within the island domain. The mostlikely location of the crosslinking site inDcPSKR1-∆KD-His6 is the region Glu503-Lys517
(Fig. 2C).
On-column photoaffinity labeling of DcPSKR1-KD-His6––To confirm the location of the
crosslinking site of ASA-PSK by massspectrometry, we performed large-scalephotoaffinity labeling followed by trypsindigestion. The main difficulty in identifyingphotoaffinity-labeled peptide fragmentscontained in the complex enzymatic digests ofthe labeled protein is that the relative abundanceof the labeled fragment is extremely low due tothe low efficiency of the photoaffinity cross-linking reaction. To overcome this limitation, weestablished a novel “on-column photoaffinitylabeling” methodology that allows repeatedincorporation of the photoaffinity label. Weimmobilized DcPSKR1-∆KD-His6 on Ni-chelating HiTrapTM HP Sepharose beads using aHis6 tag, and performed solid-phasephotoaffinity labeling in a transparent narrowcolumn by directly irradiating the column withUV light (365 nm). The crucial advantage of theon-column photoaffinity labeling is that thecross-linking reaction can be repeated afterwashing out the un-cross-linked ligands that actas potential competitors of the newly addedligands in the next round of photoaffinityreaction. In addition, this system allows directbuffer exchange without dialysis uponenzymatic digestion.
We repeated sequential on-columnphotoaffinity labeling 3 times, and confirmed thesignificant increase in cross-linking of
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[125I]ASA-PSK to immobilized DcPSKR1-∆KD-His6 (Fig. 3A). We also confirmed that labeledDcPSKR1-∆KD-His6 can be successfullydigested by trypsin even if it is immobilized onNi-chelating beads (Fig. 3A). Released labeledfragment was no longer detectable on SDS-PAGE after trypsin digestion, confirming that itsmolecular size is <3.5 kDa.
To gain more information about themolecular size of the labeled tryptic fragment,we chromatographed the tryptic digest of labeledDcPSKR1-∆KD-His6 on a gel-filtration column.Auto well gamma counting of each fractionrevealed the presence of two clear peaks (Fig.3B). HPLC analysis of the larger peak fractionshowed that radioactivity was not retained on thereverse-phase column, suggesting that theradioactive molecule contained in this fractionwas free 125I rather than labeled fragments (datanot shown). Based on the separation range ofSephadex G-25 (MW 5000 - MW 800) and theelution profiles of blue dextran (marker for V0)and cyanocobalamine (marker for molecularmass separation, MW 1355), the apparentmolecular size of the labeled peptide containedin the smaller peak was estimated to be between1.5 kDa and 5 kDa. Together with the data fromthe SDS-PAGE of the labeled tryptic peptide,we estimated the apparent molecular size of thelabeled fragment to be between 1.5 kDa and 3.5kDa.
Immuno-precipitation and MALDI-TOF MSanalysis of the labeled peptide––To directlydetect the peptide fragment cross-linked withASA-PSK using MALDI-TOF MS, we purifiedthe labeled fragment by immuno-precipitationusing anti-PSK antibodies. We performed large-scale on-column photoaffinity labeling of ≈200pmol of purified DcPSKR1-∆KD-His6 usingnon-radioactive ASA-PSK (repeated 10 times),and purified labeled fragments derived from thetryptic digest of the labeled protein using theimmuno-affinity column. Using positive-modeMALDI-TOF MS analysis, we identified twospecific molecular ion peaks at m/z 1752.83 and1880.87 in the eluate of the immuno-affinitycolumn; these peaks were not detected in thecontrol experiments using the tryptic digest ofthe unlabeled protein (Fig. 4A). Uponassignment of these peaks, we considered twopossibilities: i) that cross-linked peptidefragments were detected as an adducted form; ii)that cross-linked peptide fragments weredetected in their free form due to the loss of thephoto-incorporated moiety. MALDI-TOF MSanalysis often causes cleavage of unstable cross-linked sites. For example, MALDI-TOF MS
analysis of the purified labeled fragment ofporcine guanylyl cyclase C yielded aconsiderable amount of the free form due tocleavage of the unstable nitrene-mediated cross-linked site (22). Similar results have beenreported in studies in which ligand-bindingdomains were examined using photoaffinitylabeling (23,24).
Of the two above-described molecular ionpeaks, the peak at m/z 1752.83 perfectlymatched the calculated mass value for the freeform of the tryptic peptide fragment (Glu503-Lys517) of DcPSKR1-∆KD-His6 (calculated exactmass of ENAVEEPSPDFPFFK [in protonatedform], 1752.80) (Fig. 4B). This domaincorresponds to N-terminal side of the 36-amino-acid island domain of DcPSKR1. Similarly, thepeak at m/z 1880.87 matched the miscleavedtryptic peptide fragment (Glu503-Lys518) derivedfrom the same region of DcPSKR1-∆KD-His6
(calculated exact mass ofENAVEEPSPDFPFFKK [in protonated form],1880.89) (Fig. 4B). Because the syntheticpeptide ENAVEEPSPDFPFFK alone exhibitedno interaction with the immuno-affinity column(data not shown), it is likely that these fragmentswere immuno-precipitated with the cross-linkedASA-PSK and were generated by the cleavageof cross-linked sites presumably due to the highenergy from the laser beam during MALDI-TOFMS analysis. This possibility is supported by thefollowing three observations: i) Negative-modeMALDI-TOF MS of the same sample detectedan ion peak at m/z 900.36, which corresponds tothe [M–H–SO3]
– ion of the cleaved ligand (Fig.4A). ii) The appearance of the peak at m/z1880.87, which corresponds to the miscleavedtryptic peptide fragment (Glu503-Lys518), isconsistent with Lys517 being a cross-linking sitethat is resistant to trypsin digestion due tomodification of its side chain. iii) The calculatedmass of the adduct form (2741.10) is within theestimated range of the molecular size obtainedfrom gel-filtration experiments. Becausenegatively charged peptides such asphosphorylated and sulfated peptides are oftenresistant to ionization in positive-mode MALDI-TOF MS analysis, it is possible that the level ofionization of the adduct form was below thelimit of detection.
Deletion of Glu503-Lys517 of DcPSKR1 abolishesligand-binding activity––To confirm that the 15-amino-acid Glu503-Lys517 region within the islanddomain of DcPSKR1 is involved in ligandbinding, we generated a deletion mutant ofDcPSKR1 that lacks Glu503-Lys517 (DcPSKR1-∆ID[Glu503-Lys517]) (Fig. 5A). The ligand-
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binding assay using [3H]PSK showed that PSK-binding activity in DcPSKR1-∆ID[Glu503-Lys517]membranes decreased to background levels inspite of successful expression of truncatedproteins detected by immunoblot analysis,indicating that the region Glu503-Lys517 isnecessary for ligand binding (Fig. 5B and 5C).We also prepared deletion mutant of DcPSKR1lacking Lys518-Ile538, which is a region adjacentto Glu503-Lys517 within the island domain. Theligand-binding assay showed that DcPSKR1-∆ID[Lys518-Ile538] also completely lacked PSK-binding activity, suggesting that the entire islanddomain is necessary for ligand recognition.
We determined that the ligand-contactdomain of DcPSKR1 is located within the 15-amino-acid Glu503-Lys517 region of the islanddomain, and that Glu503-Lys517 and severaladjacent residues together form a functionalligand-binding pocket.
[Discussion]
Based on the present results of on-columnphotoaffinity labeling, MALDI-TOF MSanalysis, and fragmentation of labeledDcPSKR1-∆KD-His6 by CNBr andendoproteinase Asp-N, we conclude that the 15-amino-acid Glu503-Lys517 region located on theN-terminal side of the 36-amino-acid islanddomain is the ligand-contact domain ofDcPSKR1. This region is also highly conservedin Arabidopsis PSK receptor AtPSKR1 (10). On-column photoaffinity labeling allows repeatedcross-linking, thus increasing labeling efficiencywithout interference by un-cross-linkedphotolyzed ligands, which act as potentialcompetitors of the newly added photoaffinityligands in the next round of the photoaffinityreaction. This system is also compatible withenzymatic digestion after changing to theappropriate buffer, and enables us to useMALDI-TOF MS to directly analyze labeledfragments derived from small quantities ofnatural receptor preparations.
It has been reported that the brassinosteroid(BR) receptor BRI1, which also belongs to theLRR-RK family, directly interacts withphotoactivatable BRs, and that the 90-amino-acid region containing the island domain and anadjacent single leucine-rich repeat (LRR) canrecognize BRs even when they are bacteriallyexpressed as a glutathione-S-transferase (GST)fusion (25). These findings indicate that thisregion can, on its own, form a functional bindingpocket for recognition of BRs. However, in thepresent study, recombinant GST-fusion proteinscontaining the island domain of DcPSKR1 and
several adjacent LRRs did not bind to PSK (datanot shown), suggesting that this domain is notsufficient for formation of a stable ligand-binding pocket. The 70-amino-acid islanddomain of BRI1 contains two cysteine residues,which may form a disulfide bond that stabilizesthe ligand-binding pocket. In contrast, there areno cysteine residues within the 36-amino-acidisland domain of DcPSKR1 or the several LRRsadjacent to it. Although further experiments arerequired to demonstrate how extracellular 21tandem LRRs of DcPSKR1 contribute to theformation of a stable ligand-binding pocket, wespeculate that the global ternary structure of theDcPSKR1 extracellular domain, rather than alocal sequence motif within the island domain,defines the specific conformation of the islanddomain by which DcPSKR1 recognizes PSKwith high affinity and specificity.
The theoretical structural model of Cf-9, amembrane-localized leucine-rich repeatreceptor-like protein (LRR-RLP) of tomato cells(Lycopersicon pimpinellifolium) involved in theresistance response to the fungal pathogenCladosporium fulvum , shows that its 38-amino-acid island domain forms a unique “loop-out”structure that is isolated from the adjacent LRRloops (26). Secondary structure prediction ofDcPSKR1 using the multivariate linearregression combination (MLRC) software (27)suggests that its 36-amino-acid island domainhas no characteristic structural motif such as α-helix and β-strand which are highly conserved inLRR domain (data not shown). We propose thatthe island domain acts as a flexible hinge-likeregion that modulates the relative conformationof the two surrounding LRR regions upon ligandbinding. An island domain has been found inseveral LRR-RKs including BRI1 (28) andtBRI1/SR160 (29), and in some LRR-RLPsincluding CLV2 (30), Cf-9 (31) and LeEIX (32);each of these island domains has an unique andspecific amino-acid sequence that is distinctfrom that of the highly conserved LRR motif(33).
There are also many LRR-RKs and RLPs thathave no island domain, such as FLS2 (34), Xa21(35) and TMM (36). In a recent report,photoaffinity labeling indicates that flagellin, anelicitor-active structural component of bacterialflagella, directly binds and cross-links to FLS2(37). Although the ligand-binding site of FLS2has not been identified, two lines of evidencesuggest that a LRR domain is involved in itsligand binding. First, the Arabidopsis mutantfls2-24, which carries a point mutation in one ofits 28 LRRs, completely lacks flagellin-bindingactivity (37). Second, studies indicate that in
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mammals, flagellin binds in a highly specificmanner to the extracellular LRR domain ofTLR5 (38,39). The existence of two distinctligand perception systems among plant LRR-RKs may reflect plant strategies for adaptationto dynamic environmental conditions usinglimited molecular components. Indeed, thetomato LRR-RK tBRI1/SR160 recognizes bothBRs and systemin, and the lack of competitionbetween these two ligands suggests the presenceof two distinct binding sites in tBRI1/SR160(29).
A fundamental question in the study oftransmembrane surface receptors is how ligand
binding switches the receptor signaling statebetween active and inactive states. Identificationof the ligand-binding site of DcPSKR1 is animportant step toward clarifying the regulatorymechanisms of receptor-mediated signaltransduction. The biochemically detectableinteraction between PSK and DcPSKR1 canserve as a model system for studying themolecular basis of interaction between yet-uncharacterized ligands and putative receptors.
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[Footnotes]
* This work was supported by the 21st Century Center of Excellence Program (grant no. 14COEA02),by a Grant-in-Aid for Scientific Research for Priority Areas (grant no. 14036214) and by a Grant-in-Aid for Young Scientists (A) (grant no. 18687003).
The abbreviations used are: PSK, phytosulfokine; LRR, leucine-rich repeat; LRR-RK, leucine-richrepeat receptor kinase; DcPSKR1, Daucus carota phytosulfokine receptor 1; MALDI-TOF MS, matrixassisted laser desorption ionisation time-of-flight mass spectrometry; ∆KD, delta kinase domain; ∆ID,delta island domain; ASA-PSK, Nε-[(4-azidosalicyl)Lys5]PSK; LRR-RLP, leucine-rich repeat receptor-like protein.
[Figure legends]
Fig. 1. Overexpression of DcPSKR1-His6 and DcPSKR1- KD-His6 (A) Schematic structures ofDcPSKR1 and its derivatives. DcPSKR1 is a 1021-amino-acid LRR-RK containing 21 tandem copiesof LRR, a 36-amino-acid island domain between the 17th and 18th LRR, a single transmembranedomain, and a cytoplasmic kinase domain. (B) Northern blot and Western blot of transgenic BY-2cells overexpressing DcPSKR1-His6 and DcPSKR1-∆KD-His6. For each cell line, 10 µg of total RNAwas electrophoresed, transferred to nylon membranes, and hybridized with DcPSKR1 extracellulardomain cDNA probe (lower panel). Then, 100 µg of total membrane protein from transformants andwild-type cells was separated by SDS-PAGE and subjected to Western blot analysis using purifiedantibodies recognizing the N-terminal region of the extracellular domain of DcPSKR1 (upper panel).(C) [3H]PSK binding assay of microsomal proteins prepared from individual cell lines. Eachmicrosomal protein was incubated with [3H]PSK in the absence (open bars) or presence (filled bars) of3.2 µM unlabeled PSK. (D) Scatchard plot of the [3H]PSK binding data of DcPSKR1-∆KD-His6
microsomal fractions. The dissociation constant was calculated to be 2.1 ± 0.3 nM. (E) SDS-PAGEanalysis and deglycosylation of the affinity-purified DcPSKR1-∆KD-His6. Total proteins purifiedusing a PSK-Sepharose column and hydroxyapatite column were concentrated by ultrafiltration,separated by SDS-PAGE and visualized by SYPRO red (left panel). Purified DcPSKR1-∆KD-His6
was deglycosylated by PNGase F, separated by SDS-PAGE and visualized by immunoblot analysiswith anti-DcPSKR1 antibodies (right panel).
Fig. 2. Photoaffinity labeling of DcPSKR1- KD-His6 (A) Structure of [125I]ASA-PSK. (B)Photoaffinity labeling of DcPSKR1-∆KD-His6 and fragmentations of labeled proteins. PurifiedDcPSKR1-∆KD-His6 was incubated with 10 nM [125I]ASA-PSK in the absence (-PSK) or presence(+PSK) of excess unlabeled PSK, and was then irradiated with UV light (365 nm). Cross-linkedproteins were analyzed by SDS-PAGE and autoradiography. Fragmentations of labeled proteins by
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CNBr, Asp-N and TPCK-treated trypsin were analyzed by SDS-PAGE and autoradiography. Thedecrease in apparent molecular size after PNGase F treatment indicates the presence of N-linkedglycans. (C) Theoretical digestion map of DcPSKR1-∆KD-His6 after treatment with CNBr, Asp-N andtrypsin. Lengths of fragments are proportional to the number of amino acids. Highlighted bars withmolecular weights indicate the possible fragments labeled by the photoaffinity ligand. Met-Thrsequences are resistant to CNBr treatment due to the conversion of Met to homoserine withoutcleavage.
Fig. 3. On-column photoaffinity labeling and digestion (A) Affinity-purified DcPSKR1-∆KD-His6
(1 ml) was immobilized on Ni2+-loaded HiTrap chelating HP Sepharose. On-column photoaffinitylabeling cycle was then repeated 3 times. Aliquots of the Sepharose with labeled proteins were directlymixed with SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. The same quantityof DcPSKR1-∆KD-His6 labeled with [125I]ASA-PSK by in-solution photoaffinity labeling was loadedas a control sample (left panel). Aliquots of the labeled proteins were further on-column digested bytrypsin and analyzed by SDS-PAGE and autoradiography, confirming that the labeled band hadcompletely disappeared (right panel). (B) Gel filtration profile of tryptic digest of on-column-labeledDcPSKR1-∆KD-His6. On-column tryptic digests of [ 125I]ASA-PSK-labeled DcPSKR1-∆KD-His6 wereapplied to the Sephadex G-25 column, and 1.0-ml fractions were analyzed for radioactivity using anauto well gamma system. Fractions V0 and V t (open arrows) were identified based on the elutionvolume of blue dextran and free Na125I, respectively (data not shown). Cyanocobalamine (MW 1355),used as a marker for molecular mass separation (open arrow), was detected based on absorbance at548 nm.
Fig. 4. MALDI-TOF MS analysis of tryptic-digested on-column-labeled DcPSKR1- KD-His6 (A)Labeled DcPSKR1-∆KD-His6 fragments purified using the immunoaffinity column were analyzed byMALDI-TOF MS. The control experiment was performed using the tryptic digest of the unlabeledproteins. Specific ion peaks were detected in both the positive and negative mode. (B) Photolabeledpeptides that were detected by MALDI-TOF MS are summarized.
Fig. 5. PSK-binding activity of DcPSKR1- ID[Glu503-Lys517] and DcPSKR1- ID[Lys518-Ile538] (A)Schematic structures of DcPSKR1 and its deletion mutants lacking Glu503-Lys517 or Lys518-Ile538 withinisland domain. (B) Western blot of transgenic BY-2 cells overexpressing full-length DcPSKR1,DcPSKR1-∆ID[Glu503-Lys517] and DcPSKR1-∆ID[Lys518-Ile538]. Aliquots (100 µg) of total membraneprotein from transformants and wild-type BY-2 cells were subjected to SDS-PAGE and Western blotanalysis using purified antibodies that recognize the N-terminal region of the extracellular domain ofDcPSKR1. (C) [3H]PSK binding assay of microsomal proteins prepared from individual cell lines.Each microsomal protein was incubated with [3H]PSK in the absence (open bars) or presence (filledbars) of 3.2 µM unlabeled PSK.
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Hidefumi Shinohara, Mari Ogawa, Youji Sakagami and Yoshikatsu Matsubayashiphotoaffinity labeling
Identification of ligand binding site of phytosulfokine receptor by on-column
published online November 8, 2006J. Biol. Chem.
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