Common Mechanism of Ligand Recognition by Group II/III WW Domains
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m404719200
ISSN1083-351X
AutoresYusuke Kato, Koji Nagata, Mihoko Takahashi, Lubing Lian, Juan J. Herrero, Marius Sudol, Masaru Tanokura,
Tópico(s)14-3-3 protein interactions
ResumoWW domain is a well known protein module that mediates protein to protein interactions by binding to proline-containing ligands. Based on the ligand predilections, the WW domains have been classified into four major groups. Group II and III WW domains have been reported to bind the proline-leucine and proline-arginine motifs, respectively. In the present study, using surface plasmon resonance technique we have shown that these WW domains have almost indistinguishable ligand preferences and kinetic properties. Hence, we propose that Group II and III WW domains should be joined together as one group (Group II/III). Unlike Group I and IV WW domains, Group II/III WW domains can bind simple polyprolines as well as the proline-leucine and proline-arginine motifs, and they possess two Xaa-proline (where Xaa is any amino acid) binding grooves similar to SH3 domains. Our work assigns Group II and III WW domains to a larger family of polyproline-binding modules and proteins, which includes SH3 domains and profilin. Because polyprolines belong to the most frequently found peptide motifs in several genomes, our study implies the versatile importance of Group II/III WW domains in signaling. WW domain is a well known protein module that mediates protein to protein interactions by binding to proline-containing ligands. Based on the ligand predilections, the WW domains have been classified into four major groups. Group II and III WW domains have been reported to bind the proline-leucine and proline-arginine motifs, respectively. In the present study, using surface plasmon resonance technique we have shown that these WW domains have almost indistinguishable ligand preferences and kinetic properties. Hence, we propose that Group II and III WW domains should be joined together as one group (Group II/III). Unlike Group I and IV WW domains, Group II/III WW domains can bind simple polyprolines as well as the proline-leucine and proline-arginine motifs, and they possess two Xaa-proline (where Xaa is any amino acid) binding grooves similar to SH3 domains. Our work assigns Group II and III WW domains to a larger family of polyproline-binding modules and proteins, which includes SH3 domains and profilin. Because polyprolines belong to the most frequently found peptide motifs in several genomes, our study implies the versatile importance of Group II/III WW domains in signaling. The WW domain is composed of 30–40 amino acids and named after two tryptophan (W) residues that are highly conserved and spaced 20–22 amino acids apart (1Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Google Scholar, 2Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Google Scholar). The domain binds proline-rich or proline-containing ligands (3Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Google Scholar, 4Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Google Scholar, 5Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Google Scholar). WW domain-containing proteins have been shown to be involved in a variety of cellular processes including cell cycle control (Pin1/Ess1), ubiquitin ligation (Nedd4/Rsp5 and smurf1), and coactivation of transcription (YAP65) (reviewed in Refs. 2Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Google Scholar and 6Kay B.K. Williamson M.P. Sudol M. FASEB J. 2000; 14: 231-241Scopus (1026) Google Scholar). A few WW domain-containing proteins have been implicated, either directly or indirectly, in a variety of human diseases such as Liddle's syndrome of hypertension, Duchenne and Becker muscular dystrophies, Huntington's disease, and Alzheimer's disease (7Faber P.W. Barnes G.T. Srinidhi J. Chen J. Gusella J.F. MacDonald M.E. Hum. Mol. Genet. 1998; 7: 1463-1474Google Scholar, 8Huang X. Poy F. Zhang R. Joachimiak A. Sudol M. Eck M.J. Nat. Struct. Biol. 2000; 7: 634-638Google Scholar, 9Sudol M. Sliwa K. Russo T. FEBS Lett. 2001; 490: 190-195Google Scholar). FBP11, a mammalian homologue of yeast Prp40, binds the Pro-Leu (PL) motif (4Chan D.C. Bedford M.T. Leder P. EMBO J. 1996; 15: 1045-1054Google Scholar). FBP11 and WAC act as a component of the splicing factor (10Bedford M.T. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Google Scholar, 11Xu G.M. Arnaout M.A. Genomics. 2002; 79: 87-94Google Scholar). FBP11, HYPB, and HYPC also bind Huntingtin, a protein responsible for Huntington's disease (7Faber P.W. Barnes G.T. Srinidhi J. Chen J. Gusella J.F. MacDonald M.E. Hum. Mol. Genet. 1998; 7: 1463-1474Google Scholar). Fe65 binds β-amyloid precursor protein, a precursor of β-amyloid peptide, which constitutes the extracellular neuritic plaques in Alzheimer disease (9Sudol M. Sliwa K. Russo T. FEBS Lett. 2001; 490: 190-195Google Scholar, 12Selkoe D.J. Science. 1997; 275: 630-631Google Scholar). The WW domain of Fe65 binds the proline-rich region of Mena, a mammalian homologue of Drosophila melanogaster Ena, which has been identified in a specific screen for dominant mutations that alleviate the Abl phenotype (13Gertler F.B. Doctor J.S. Hoffmann F.M. Science. 1990; 248: 857-860Google Scholar, 14Ermekova K.S. Zambrano N. Linn H. Minopoli G. Gertler F. Russo T. Sudol M. J. Biol. Chem. 1997; 272: 32869-32877Google Scholar). Fe65 and β-amyloid precursor protein are involved in the reconstruction of actin cytoskeleton, which suggests the concerted action of these proteins with Mena and/or Abl (9Sudol M. Sliwa K. Russo T. FEBS Lett. 2001; 490: 190-195Google Scholar, 15Sabo S.L. Ikin A.F. Buxbaum J.D. Greengard P. J. Cell Biol. 2001; 153: 1403-1414Google Scholar). The WW domain of FBP30 has been reported to bind the Pro-Arg (PR) motif (10Bedford M.T. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Google Scholar, 16Bedford M.T. Sarbassova D. Xu J. Leder P. Yaffe M.B. J. Biol. Chem. 2000; 275: 10359-10396Google Scholar). WW domains have been classified into four groups according to their ligand specificity: Group I recognizes Pro-Pro-Xaa-Tyr (PY motif); Group II recognizes Pro-Pro-Leu-Pro (PL motif); Group III recognizes proline-rich segments with Arg residues (PR motif); and Group IV recognizes Ser(P)/Thr(P)-Pro-(pS/pT-P motif) (2Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Google Scholar, 9Sudol M. Sliwa K. Russo T. FEBS Lett. 2001; 490: 190-195Google Scholar). According to this classification, the WW domains of FBP11 and Fe65 have been assigned to Group II, whereas that of FBP30 has been assigned to Group III. The crystal structures of the Group I and IV WW domains complexed with their ligands have revealed a common structural basis to recognize the Pro-containing ligands; the XP groove, which is formed by two aromatic rings with nearly parallel alignment, plays a pivotal role in recognizing the Xaa-Pro segment in the ligands (17Zarrinpar A. Lim W.A. Nat. Struct. Biol. 2000; 7: 611-613Google Scholar, 18Macias M.J. Wiesner S. Sudol M. FEBS Lett. 2002; 513: 30-37Google Scholar, 19Zarrinpar A. Bhattacharyya R.P. Lim W.A. Science STKE. 2003; 179: 1-10Google Scholar). In addition, Group I and IV WW domains have been shown to possess their respective characteristic ligand recognition sites, the Tyr-binding groove (Tyr groove), and the phosphate-binding patch (“p” patch), which contribute to the specific ligand recognition (20Kato Y. Ito M. Kawai K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 10173-10177Google Scholar). However, the classification between the Group II and III WW domains has been nebulous. For instance, the WW domain of Fe65 has been classified as Group II in one report (14Ermekova K.S. Zambrano N. Linn H. Minopoli G. Gertler F. Russo T. Sudol M. J. Biol. Chem. 1997; 272: 32869-32877Google Scholar) but as Group III in another report (16Bedford M.T. Sarbassova D. Xu J. Leder P. Yaffe M.B. J. Biol. Chem. 2000; 275: 10359-10396Google Scholar). In addition, there are a few proposed ligand motifs that can also bind some of the WW domains in these classes: the PGR motif and polyprolines (7Faber P.W. Barnes G.T. Srinidhi J. Chen J. Gusella J.F. MacDonald M.E. Hum. Mol. Genet. 1998; 7: 1463-1474Google Scholar, 10Bedford M.T. Reed R. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10602-10607Google Scholar, 21Komuro A. Saeki M. Kato S. J. Biol. Chem. 1999; 274: 36513-36519Google Scholar, 22Bedford M.T. Frankel A. Yaffe M.B. Clarke S. Leder P. Richard S. J. Biol. Chem. 2000; 275: 16030-16036Google Scholar). To examine the functional classification of the Group II and III WW domains in detail, we have performed quantitative binding experiments using surface plasmon resonance (SPR). 1The abbreviations used are: SPR, surface plasmon resonance; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PPII, polyproline type II. 1The abbreviations used are: SPR, surface plasmon resonance; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PPII, polyproline type II. We also analyzed binding data generated by proteomic mapping of the WW domain (23Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M Rodwell J Herrero J.J. Proteomics. 2004; 4: 643-655Google Scholar). We found that the WW domains previously classified as Groups II and III showed very similar ligand preference and that they were able to bind each other's cognate ligands. We therefore propose that the Group II and III WW domains should be classified into one group, Group II/III. Moreover, we have performed molecular modeling and revealed that both the Group II and III WW domains commonly have a characteristic groove that is formed by two aromatic rings with nearly parallel alignment, very similar to the XP groove, on the ligand-binding surface. The importance of this characteristic second XP groove (which we named “XP2”) to recognize their ligands was confirmed by site-directed mutagenesis. Our report redefines classification of WW domains and provides an explanation for the molecular mechanisms behind this new classification. Plasmid Construction—The DNAs encoding the WW domains of human Pin1 and Caenorhabditis elegans Y110A2AL.13 gene product (Y110) were synthesized and amplified by a modified PCR in which several synthetic oligonucleotides were used as the template for the first cycle. The DNA encoding the second WW domain of Saccharomyces cerevisiae Rsp5 (Rsp5p(WW2)), human Fe65L2, and the first WW domain of mouse FBP30 (FBP30A) were amplified by PCR using their cDNAs as templates, which were kindly provided by Dr. H. Tanahashi and Dr. M. T. Bedford (16Bedford M.T. Sarbassova D. Xu J. Leder P. Yaffe M.B. J. Biol. Chem. 2000; 275: 10359-10396Google Scholar, 24Tanahashi H. Tabira T. Biochem. Biophys. Res. Commun. 1999; 258: 385-389Google Scholar). The PCR products were inserted between the BamHI and EcoRI sites of the expression vector pGEX-4T-1 (Amersham Biosciences). The expression plasmids for GST fusion proteins of the third WW domain of mouse Nedd4 (mNedd4(WW3)) and the first WW domain of human YAP65 (hYAP65(WW1)) were kindly provided by Dr. C.-K. J. Shen (25Gavva N.R. Gavva R. Ermekova K. Sudol M. Shen C.-K.J. J. Biol. Chem. 1997; 272: 24105-24108Google Scholar). The expression plasmid for GST fusion protein of the first WW domain of mouse FBP11 (FBP11A) was kindly provided by Dr. M. T. Bedford (26Bedford M.T. Chan D.C. Leder P. EMBO J. 1997; 16: 2376-2383Google Scholar). The WW domains of HYPB, WAC, and Fe65 were inserted into pGEX-4T-2 vectors as described earlier by Hu et al. (23Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M Rodwell J Herrero J.J. Proteomics. 2004; 4: 643-655Google Scholar). Expression and Purification—All the GST fusion WW domains were expressed in Escherichia coli BL21(DE3) or BL21(DE3)/pLysS (Novagen) at 37 °C and purified with glutathione-Sepharose (Amersham Biosciences). The GST tag was removed by thrombin (Sigma) digestion at 4 °C for the SPR analysis. The WW domains were separated from GST and thrombin by reverse phase chromatography with a Resource RPC 1-ml column (Amersham Biosciences) with a linear gradient of 1–40% acetonitrile (1 ml/min, 20 min) in 20 mm ammonium formate, pH 7.0. Their molecular masses were verified by MALDI-TOF mass spectrometry on a Voyager mass spectrometer (Applied Biosystems). The samples used in the proteomic mapping were prepared as described (23Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M Rodwell J Herrero J.J. Proteomics. 2004; 4: 643-655Google Scholar). Proteomic Mapping—Purified GST fusions of human WW domains were plated on 96-well plates. Employing automated equipment, complete cross-affinity matrices were generated between GST-WW fusions and proline-rich peptides derived from the human proteome. Peptidestreptavidin/alkaline phosphatase reaction was used to monitor the relative strength of binding. Quality controls and checks were employed as previously described (23Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M Rodwell J Herrero J.J. Proteomics. 2004; 4: 643-655Google Scholar). Peptide Synthesis for the SPR Analysis—Pro-containing ligand peptides were synthesized by solid phase peptide synthesis on a PSSM8 Peptide Synthesizer (Shimadzu, Kyoto, Japan). Rink amide AM resin (Novabiochem) and Fmoc-amino acids with protected side chains were used (Novabiochem). For phosphoserine and phosphothreonine, Fmoc-Ser(PO(OBzl)OH)-OH and Fmoc-Thr(PO(OBzl)OH)-OH (Novabiochem) were used, respectively. Synthesized peptides were cleaved from the resin and deprotected in trifluoroacetate in the presence of scavengers (5% water, 5% thioanisole, 3% ethylmethylsulfide, 2.5% ethandithiol, 2% thiophenol) and were then purified by reverse phase chromatography with a column ODS-AP303 (4.6 × 250 mm; YMC, Kyoto, Japan). Their molecular masses were verified by MALDI-TOF mass spectrometry on a Voyager mass spectrometer (Applied Biosystems). The amino acid sequences of the ligand peptides were GTPPPPYTVG (WBP1 peptide containing the PY motif), SPPAPPTPPPLPPP (formin peptide containing the PL motif, mouse), PPPPPPPPPPPPPP (14-meric polyproline called PP in the present paper), CGGGPPGPPPRGPPPR (WBP11 peptide containing the PR motif), GGVPRpTPV (Cdc25c peptide containing the pS/pT-P motif), and KGGPPApTPP (Myt1 peptide containing the pS/pT-P motif). SPR Binding Assay—We measured SPR using a BIAcore 2000 (Biacore). Each ligand peptide was immobilized on a flow cell of a sensor chip CM5 (Biacore) by the standard 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride/N-hydroxysuccinimide method with a 1 mg/ml peptide solution dissolved in 25 mm NaHCO3. For reference SPR signals, 25 mm NaHCO3 was used instead of a peptide solution in the immobilization reaction. The remaining activated esters were inactivated with ethanolamine. The binding experiments were performed at 20 °C using the WW domains dissolved as analytes in 20 mm HEPES, pH 7.0, 100 mm NaCl, and 0.5 mm EDTA. The flow cells were regenerated with 100 mm NaOH. The concentrations of the WW domains were determined based on the absorbance at 280 nm of the protein solutions. The dissociation constants (KD) were determined by Scatchard plot analysis (27Lemmon M.A. Ladbury J.E. Mandiyan V. Zhou M. Schlessinger J. J. Biol. Chem. 1994; 269: 31653-31658Google Scholar, 28Oda M. Furukawa K. Sarai A. Nakamura H. FEBS Lett. 1999; 454: 288-292Google Scholar). First, the raw sensorgrams of a flow cell without an immobilized ligand peptide were subtracted from the raw sensorgrams of ligand-bound flow cells. Then RUeq/C values were plotted against the RUeq values, where RUeq was the SPR response under the equilibrium, and C is the millimolar concentration of the analyte. Plotted data were fit to the equation: RUeq/C = -1/KD × RUeq + A (a constant), to determine KD. All the R2 values for the fittings by linear functions were >0.91. The association and dissociation rate constants (kon and koff) were determined from the direct fitting method using BIAevaluation 2.1. Molecular Modeling—Homology modeling was carried out using Swiss-Model (29Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar), an automated protein-modeling server combining homology search, model building, and energy minimization. The crystal structures of Pin1 (Protein Data Bank code 1PIN), and dystrophin (Protein Data Bank code 1EG3) and the NMR structures of FBP28 (Protein Data Bank code 1E0L) and mNedd4(WW3) (Protein Data Bank code 1I5H) were used as the templates for modeling the WW domains of FBP11A, Fe65L2, and FBP30A. Molecular surfaces and electrostatic potentials were calculated and displayed using MOL-MOL (30Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Google Scholar). The Swiss PDB Viewer was used to draw the backbone model (29Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar). SPR Binding Assay—We performed quantitative binding experiments by SPR to evaluate the ligand specificity of several WW domains belonging to Groups I-IV. Examples of SPR assay and analysis are shown in Fig. 1, A and B. The polypeptide ligands used were WBP1, Formin, WBP11, and Cdc25c/Myt1, which contain the PY, PL, PR, and pS/pT-P motifs and are the typical ligands for Group I, II, III, and IV WW domains, respectively. In addition, 14-meric polyproline (called PP in the present paper) was used. Eleven different WW domains were used as binding probes: the WW domains of hYAP65, Rsp5p, and mNedd4 (previously classified as Group I); those of FBP11A and HYPB (previously Group II); that of FBP30A (previously Group III); and those of Pin1 and Y110 (previously Group IV) (20Kato Y. Ito M. Kawai K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 10173-10177Google Scholar). The WW domains of WAC and Fe65L2 have never been experimentally classified before. None of the WW domains used in the present SPR analysis was in the form of the GST fusion. The GST fusion proteins have the propensity to form dimers because of the nature of the GST component. This activity could cause the problem of avidity in the measurements of SPR. Thus, the proteins we used in the present study were digested and separated from the GST before the measurement. SPR Binding Assay of WW Domains Previously Classified as Group I—Many proteins have multiple WW domains. In our SPR experiments, we used only individual WW domains from such multi-WW domain proteins. The first WW domain of hYAP65 (hYAP65 (WW1)), the second WW domain of Rsp5 (Rsp5p(WW2)), and the third WW domain of mNedd4 (mNedd4(WW3)) interacted most strongly with the WBP1 peptide that contained a PY motif, the consensus sequence for the Group I ligands, with KD values of 11–71 μm (Fig. 1C). These values are in good agreement with the KD values determined for Group I WW domains using various techniques including isothermal titration microcalorimetry (31Macias M.J. Hyvonen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Google Scholar, 32Linn H. Ermekova K.S. Rentschler S. Sparks A.B. Kay B.K. Sudol M. Biol. Chem. 1997; 378: 531-537Google Scholar). Considering these results, the problem of avidity was well avoided in the present measurement system. The WW domains of hYAP65(WW1) and rsp5p(WW2) also bound to the formin peptide that contains a PL motif, a consensus sequence for the Group II ligands. However, their bindings to the formin peptide were 19 and 50 times weaker in KD than those to the WBP1 peptide. These data show that the WW domains of hYAP65(WW1), rsp5p(WW2), and mNedd4(WW3) bound highly specifically to the PY motif, the Group I ligand. None of the Group I WW domains bound to PP peptide. SPR Binding Assay of WW Domains Previously Classified as Group II—The binding preference to the PP peptide were shown for the WW domains of FBP11A, HYPB, WAC, Fe65, and Fe65L2 (Fig. 1C). Moreover, the PL and PR motifs exhibited similar binding strength to those WW domains. Thus, a broader range in binding specificity seems common for Group II WW domains. For the WW domain of FBP11A (Group II), it was quantitatively confirmed that the most preferable ligand was the formin peptide, the PL motif. The PP and WBP11 peptide (the PR motif) also bound to FBP11A with only 2.1 and 3.3 times larger KD values, respectively, than the PL motif. The WW domain of WAC proved to possess the Group II specificity because it bound to the PP peptide most effectively. In addition, its relative binding abilities to the other Group II and III ligands were similar, with at most 2.1 times larger KD values. As was the case with the WAC WW domain, the WW domains of HYPB, Fe65, and Fe65L2 bound most strongly to PP in our SPR experiments. The WW domains of WAC, Fe65 and Fe65L2 bound also to the PL and PR motif at most with 8.4 and 2.5 times larger KD values than to the PP peptide, respectively. On the other hand, the WW domain of HYPB showed preference to the PP peptide, to which HYPB bound 19 and 20 times more strongly than to the PL and PR motifs, respectively. It is noteworthy that the WW domain of HYPB bound to the PL and PR motifs with similar KD values. The similar binding strength to both the PL and PR motifs was also observed in the other Group II WW domains (FBP11A, WAC, Fe65, and Fe65L2) reported here, because the differences of KD values were at most only 4.1 times. The controversial group classification of Fe65 was clarified in the present report. Our results showed that Fe65 best preferred the PP peptide, the Group II ligand, but also bound to the PR motif, the Group III ligand, with the small difference of KD values (2.5 times). We should consider the PP peptide as one of the general ligand motifs for the WW domains because those of HYPB, Fe65, and Fe65L2 showed significantly stronger binding to PP than to the PL motif. SPR Binding Assay of WW Domains Previously Classified as Group III—The WW domain of FBP30A, a Group III WW domain, expectedly bound most strongly to the PR motif, the typical Group III ligand, and unexpectedly bound to the PL and PP motifs, the Group II ligands, to a considerable extent (Fig. 1C). FBP30A bound to the PP and formin peptide with only 3.3 and 8.0 times larger KD values, respectively, than to the WBP11 peptide. It was also found that FBP30A bound to the PL motif more strongly than FBP11A, a Group II WW domain. Our data on the Group II and III WW domains clearly indicate that Group II and III WW domains share their ligands, the PL, PR, and PP motifs, and that in all cases their ligand preferences are very similar or almost indistinguishable. SPR Binding Assay of WW Domains Previously Classified as Group IV—It is confirmed here that the Group IV WW domains bind specifically to peptides containing the pS/pT-P motif. The WW domains of Pin1 and Y110 were previously classified as Group IV (2Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Google Scholar, 5Lu P.J. Zhou X.Z. Shen M. Lu K.P. Science. 1999; 283: 1325-1328Google Scholar, 20Kato Y. Ito M. Kawai K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 10173-10177Google Scholar). In the present study we confirmed that WW domain of Pin1 and Y110 bound most strongly to the cognate peptides derived from the Cdc25c and Myt1 proteins (Fig. 1C). The WW domain of Pin1 interacted weakly with the WBP11 peptide, a Group III ligand, with a KD value of 1.13 mm, i.e. 44 times less strongly than with the Cdc25c peptide. Thus, the bindings of the Group IV WW domains were highly specific to the Group IV ligands. It should be noted that none of the Group IV WW domains bound to the PP motif. Kinetics of WW Domains Previously Classified as Group II or III—The kinetic parameters of the Group II and III showed the extremely large koff values, i.e. the fast dissociation (Fig. 1D). Furthermore, the Groups II and III WW domains share common nature not only in terms of the specificity of ligand recognition but also in the binding kinetics. The kinetic parameters of ligand binding were compared for WW domains from Groups II and III (Fig. 1D). All of these WW domains showed a common tendency in the rate constants for both association and dissociation; those WW domains associated faster with the PP motif than with the PL and the PR motifs and dissociated from the PL motif faster than from the PP and PR motifs. It is noteworthy that the WW domain of HYPB associated with the PP motif extremely fast, with the rate constant of 2.2 ± 1.3 × 105m-1 s-1. This relative speed of association must be one of the underlying features of its high specificity to the PP motif. Proteomic Mapping—We also performed the proteomic mapping assay for the selected WW domains examined above and confirmed the common ligand predilection of the Group II and III WW domains. From Table I, we can see that all three domains have much lower number of more strongly binding ligands (optical density, >2.5) from the PY-containing peptides of the Group I. The percentage of more strongly binding ligands in Group I is in the range of 1–2%, whereas the percentages of more strongly binding ligands containing the PL, PP, and PR motifs are in the range of 5–8, 10–23, and 5–6%, respectively (Tables I and II). Thus, these three WW domains commonly prefer the PP motif best, the PL and PR motifs second best, and the PY motif worst. It is noteworthy that these WW domains prefer the PL (the Group II ligand) and PR (the Group III ligand) motifs to a similar extent. The data indicate that the difference in ligand preference of these three WW domains is almost indistinguishable. These results are consistent with the data from the SPR assay as described above.Table IThe binding distribution of three WW domains among three groups of Pro-containing ligands The binding profiles of three WW domains (from HYPB, WAC, and Fe65) are presented as the number (percentage) of one-group ligands that a given domain binds to in four optical density ranges. The total number of ligands tested against the given domain is also provided. The detailed background and methods used to acquire these data are as described (23Hu H. Columbus J. Zhang Y. Wu D. Lian L. Yang S. Goodwin J. Luczak C. Carter M. Chen L. James M. Davis R. Sudol M Rodwell J Herrero J.J. Proteomics. 2004; 4: 643-655Google Scholar). The ligands used in this analysis were generated by mining Swiss Prot human protein sequences with the core patterns (e.g. PPLP) through pattern matching. Specifically, a ligand is generated by matching the core pattern to a protein sequence and taking on average of 4 amino acids to both the N and C termini of the protein sequence as flanking region. The average ligand length is 12 amino acids. The criteria used to categorize a ligand into a group are for the core pattern (the PY, PL, PP, or PR motif) to be situated at least 2 amino acids from either end of a ligand. The examples of ligands used in this assay are listed in Table II. The sequences included in the respective motifs are described in the arcs. The domain numbers (D00188, D00189, and D00243) are internally generated identifiers.DomainsGroupLigand motifsTotal number of ligandsOptical density 2.5HYPB (D00188)IPY (PPXY)764648 (84.8%)84 (11.0%)16 (2.1%)16 (2.1%)IIPL (PPLP)445359 (80.7%)33 (7.4%)17 (3.8%)36 (8.1%)IIPP (PPPP)17675 (42.6%)39 (22.2%)22 (12.5%)40 (22.7%)IIIPR (PPR)405298 (73.6%)62 (15.3%)26 (6.4%)19 (4.7%)WAC (D00189)IPY (PPXY)752622 (82.7%)95 (12.6%)28 (3.7%)7 (0.9%)IIPL (PPLP)445342 (76.9%)54 (12.1%)28 (6.3%)21 (4.7%)IIPP (PPPP)17697 (55.1%)40 (22.7%)18 (10.2%)21 (11.9%)IIIPR (PPR)405314 (77.5%)49 (12.1%)17 (4.2%)25 (6.2%)Fe65 (D00243)IPY (PPXY)752554 (73.7%)153 (20.3%)35 (4.7%)10 (1.3%)IIPL (PPLP)445238 (53.5%)101 (22.7%)77 (17.3%)29 (6.5%)IIPP (PPPP)17672 (40.9%)55 (31.3%)32 (18.2%)17 (9.7%)IIIPR (PPR)405344 (84.9%)27 (6.7%)10 (2.5%)24 (5.9%) Open table in a new tab Table IIThe list of randomly selected example of ligands containing each motif for proteomic mappingPY motif ligandsPL motif ligandsPP motif ligandsPR motif ligandsPGTPPPNYDSERLWVGLPPLPSARCRFCPPPPPPYQNQSERMVPPRPDSLTSRGLPPPYDLTWVNAAPPRLLPPLPTCYGPPALPPPPPLAKFPGPPQPPPRAGPPKWEGEPPPPYSRPGVKLSPPLPPKKVMSQGPPPPPPYGRLLGLPQPPRASGQTPIAPPTYWEWALYENLPPLPPVWESPPPPPPPPPHSFIKQPAVPPRPSADLFSNRPPGYPSQPVSALEPPLPAAQSDFLPPPPPPLDDSSAPSHPPRPLLPLMSFHPPHYLAARAATMPPLPQPPLAPASGNFPPPPPLDEEAEEESPPRPSLSQLCYEPPTYSPAGPPASPPLPLPPPAAVQQQQPPPPPIPANGRKEEPPRPEFLEQTLVPPAYAPYPSITVNPPLPQDTVSLIPPPPPPKNVARLCRPPPRAPRAPAPYVEPPEYEFFWLSSFPPLPGAAGVPPPPPPPPFLRPGRAGRPPRPAAGPACWREPPAYTYRDSVMTPPLPLQDTIPAAPPPPPYPYLVYRHPPPRPANFE Open table in a new tab The GST fusion proteins of the WW domains were used in this proteomic mapping. However, the problem of avidity was probably significantly minimized because the fused WW domains were immobilized and not presented as free flowing samples in solution. Comparison of the Ligand-binding Sides of Groups I-IV WW Domains—The results of our survey prompted us to re-examine ligand-binding pockets of WW domains in hope of finding structural denominators common for Group II and III WW domains. Groups I and IV WW domains employ the same side of their bent-sheeted structure to recognize their respective ligands (Fig. 2, A–C). We assumed that the WW domains previously classified as Groups II and III would also use the same side to recognize their ligands. We constructed molecular models of the Group II and III WW domains (FBP11A, Fe65L2, and FBP30A) based on the sequence homology and compared their putative ligand-binding sides with those of Group I and IV WW domains to reveal the structural basis of their ligand specificity (Fig. 2, D–F). We have defined the common num
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