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1 Institut für Medizinische Immunologie, Universitätsklinikum Charité, Schumannstr. 20-21, 10117 Berlin, Germany
2 Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany
3 The Berlin Structure Factory Project, Heubnerweg 6, 14059 Berlin, Germany
Reprint requests to: Hartmut Oschkinat, Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Str. 10, 13125 Berlin, Germany; E-mail: Oschkinat{at}fmp-berlin.de; fax: 49 (30) 94793-169.
4 These authors contributed equally to this work. 
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0233203.
Abbreviations and conventions: Consensus sequences are given according to modified Seefeld Convention 2001 nomenclature (Aasland et al. 2002). Amino acids are represented by standard IUPAC single-character notation with uppercase bold characters representing the most highly conserved positions lowercase characters representing less specific residues lowercase "x" denoting "any amino acid," amino acid alternatives specified within brackets, 
representing hydrophobic amino acids, 
representing aliphatic amino acids, and phosphorylated amino acids denoted by a po-prefix. Multiple WW domains within one protein are sequentially abbreviated by the protein name, with the sequential number of the respective WW domain as a suffix. WW domains of proteins containing only a single WW domain are represented by the protein name only, omitting a suffix. n.d., not determined.
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Abstract |
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 TOP Abstract IntroductionResults and DiscussionMaterials and methodsReferences |
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)P(p,g)PPpR, (p/)PPRgpPp, PPLPp, (p/)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases. Keywords: Protein—protein interaction; WW domain; protein interaction modules; ligand screen; peptide libraries; sequence—activity relationships; protein—protein interaction prediction
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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We attempted to identify specificity-determining residues within WW domains and their ligand-recognition motifs. Thereby, sequence activity relationships could be inferred that then allowed for the prediction of potential activities and interaction partners of yet unexplored WW domains and ligands. These predictions aid the design of experiments to assess the cellular function of WW domain-containing proteins, and hence help to unravel the protein interaction network. As these dynamic networks have to respond to changing environments, mechanisms are required to regulate the underlying protein interactions. Hence, we also investigated the potential regulation of WW domain–ligand interaction by phosphorylation of the ligand (Sudol and Hunter 2000).
To determine the ligand binding propensities of 42 chemically synthesized WW domains (29 human, 2 mouse, 11 yeast), we used NMR spectroscopy for detecting interactions with nonoptimized model peptides in a site-specific manner. As a second, independent approach we used a cellulose-bound array of the 42 WW domains for solid-phase binding assays with peptides derived from naturally occurring proteins. To infer sequence activity relationships, the ligand binding propensities were mapped onto a tree, which clusters WW domains based on sequence similarity. Group-specific consensus sequences of the WW domains were obtained. Substitutional analyses of the ligands revealed group-specific binding patterns suggesting common group-binding mechanisms. Regulation of WW domain–ligand interactions by ligand phosphorylation was analyzed by screening a library consisting of 600 potential kinase target sequences and determination of dissociation constants by fluorescence spectroscopy. Finally, a profile hidden Markov model (HMM) was used to predict the binding propensities towards tyrosine/phospho-tyrosine-containing ligands for the remaining WW sequences found in the SMART database (Schultz et al. 1998).
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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According to the 1D-1H NMR reference spectra, without ligand, 30 WW domains were folded, showing the characteristic peak pattern between 9.0–9.6 and 10 ppm (Macias et al. 1996; Figs. 1A, 2
). Two further domains, hWWP3-2 and hPQBP1, were folded only after ligand addition, judged by the appearance of the characteristic peak pattern in the 1D-1H NMR spectra. Sixteen WW domains showed chemical shift perturbations after addition of both the Y- and the poY-peptide. For four more domains only weak perturbations were observed with the poY- but none with the Y-peptide (Fig. 2, first and second result columns). WW array experiments using tyrosine- and phospho-tyrosine-containing peptides supported the NMR results. The NMR screen for the poS-peptide revealed seven potential weak interactions, six of these with WW domains also recognizing phospho-tyrosine (Fig. 2, third result column). An additional seven domains showed chemical shift changes when exposed to a mixture of model peptides of the type GPPPPBG (where B = E, I, Q, R, and phospho-threonine [poT]).
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, fifth and sixth result columns), with the short R-peptide being more selective. Using the short and long L-peptides, respectively, two and nine positive responses were obtained (Figs. 1C, 2, seventh and eighth result columns).
In summary, binding to specific proline-containing ligands is a general feature of WW domains. In total, 31 WW domains bound to at least one of the tested proline-containing peptides. Ten of the 11 domains that did not show any potential interaction were unfolded, as shown by the 1D-1H NMR spectra. The screening results allowed a definition of six groups of WW domains according to their activity (Fig. 2): WW domains recognizing exclusively the Y- and poY-peptide constitute the Y-group. For arginine-containing ligands, two distinct groups could be identified: The Ra-group, whose members bound strongly to both R-peptides and in addition the long L-peptide, and the Rb-group showing strong interactions solely with both arginine-containing peptides. Strong binding to the short and long L-peptide was found for L-group members. Distinct from these, a group of WW domains, which bind to the long L-peptide only, was found. We called this the poly-P-group, because the long L-peptide contains a stretch of five prolines distinguishing it from the short L-peptide. The well-known phospho-serine/threonine-recognizing WW domains hPIN1 and yESS1 (Verdecia et al. 2000) were combined to form the sixth group.
Sequence activity relationships
To cluster the 42 WW domains on the basis of conserved sequence features, a tree representing sequence similarity was calculated (Felsenstein 1989; Page 1996). Only residues on the ligand-exposed face of the ß-sheet were included to bias the tree towards functional relatedness. Mapping of the detected ligand binding propensities (Fig. 2) onto this tree revealed a correlation between the Y-, Ra-, and Rb-group and three distinct branches (Fig. 3). All domains recognizing exclusively tyrosine- and phospho-tyrosine-containing peptides (Y-group) are clustered into a compact branch (Y-branch), which does not contain domains binding strongly to any other motif. hSMURF2-1, yYJQ8 and four unfolded domains, for all of which no ligands were found, are also found in the same branch. Domains outside this branch showing weak tyrosine- or phospho-tyrosine-affinity invariably bound more strongly to R-, L-, or poT-peptides. All R-, L-, and poT-specific domains are located in the lower half of the tree. In this region two distinct branches cluster Ra-group and Rb-group members, respectively. In contrast to this, the L-, poly-P-, and poS/poT-group WW domains are more dispersed in the lower half of the tree. The phylogenetic tree calculated using all residues does not differ fundamentally from the one presented here (data not shown). Most notably the Y-, Ra-, and Rb-branches are found in this tree as well. The trees differ mainly in the fact that yYJQ8 does not cluster to the Y-branch, and hHYP109-1 not to the Ra-branch.
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). The L- and poly-P-group consensus sequences were omitted, because these groups had not enough members, and thus the respective consensi were not revealing. All other residue patterns share the residues characteristic for WW domains (structural: W17, Y/F29, and P42; functional: Y28, S/T37, and W39). Beyond this the residue patterns show distinct group specific features (Figs. 2, 4). In the case of the Y-group, the consensus additionally comprises E18, V30, H32, and R35. Except for E18, these residues have been shown by the available complex structures to mediate the tyrosine recognition (Macias et al. 1996). In the case of the two different R-groups, two distinct group-specific residue patterns were found: V20, D22, and W30 for the Ra-group and V18, G20, T/S22, D/E24, Y/F26, and Y30 for the Rb-group. Despite their differences, negatively charged residues in the ß1–ß2-loop in conjunction with an aromatic residue in position 30 are characteristic for both consensus sequences. These negatively charged residues might recognize the arginine of the ligand. Their location in the ß1–ß2-loop closely resembles the location of the S22R/K23-motif of the poS/poT-group members recognizing the poS/poT residues in their ligands, suggesting an analogous ligand recognition mechanism. Contrary to this, the specificity-determining residues of the Y-group are mainly located around the ß2–ß3-loop. The presence of a third aromatic residue on the binding surface of the WW domains of both R-groups might lead to the formation of a second proline binding site (Macias et al. 2002). This second proline binding site might be even more pronounced in the Ra-group by the presence of a tryptophan, which may explain their propensity to bind longer proline only sequences (Fig. 2, eighth result column). In fact, hFE65 has already been reported to bind to the peptide PPPPPP (Espanel and Sudol 1999).
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Analysis of group-specific binding motifs
To identify the key residues of the ligand and to address the question of whether our classification shows group-specific binding motifs, substitutional analyses of the identified ligands were performed for all members of the Ra-, Rb-, L-, poly-P-, and poS/poT-group and for selected and predicted members of the Y-group.
In the case of the Y-group substitutional analyses of the respective ligands are already known for several members, for example, hYAP65 (Pires et al. 2001). The characteristic substitutional pattern could be reproduced for selected members of the Y-group and for WW domains predicted to be Y-group members (see below), like hBAG3 shown in Figure 5A. The residues P-3, P-2, and Y0 are the key residues recognized by WW domains of the Y-group (Aasland et al. 2002). The substitutions tolerated at positions -1 might be domain- but not group-specific. An amino acid in position -4 is not essential but necessary for binding with higher affinity, as shown by length analyses. Altogether, the conserved binding motif for the Y-group can be summarized as PPxY.
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, B and C, respectively. In the case of the Ra-group P-5, P-3, P-2, and R0 were found to be the key residues, which could not be substituted at all (Fig. 5B). Positions -4 and -1 were less restricted, but showed a clear preference for glycine or proline, in case of position -4, and proline, in the case of position -1. Although still quite specific, P-6 could be replaced by hydrophobic residues. The four C-terminal positions seemed not to be important for recognition, which was confirmed by length analyses (data not shown). Therefore, the preferred binding motif for the Ra-group can be summarized as (p/)P(p,g)PPpR.
In the case of the Rb-group the key residues comprise P-2, P-1, R0, G1, and P3 (Fig. 5C). P-3, P2, and P4 could be replaced by other amino acids, albeit resulting in lower binding affinity. Substitution of the 3 N-terminal positions by different amino acids had only minor effects on the binding affinity. Together this results in (p/)PPRgpPp as the preferred binding motif for the Rb-group.
The main difference between the Ra- and Rb-group binding patterns is the relative position of the key residues with respect to the arginine. In the case of the Rb-group both N-terminal and C-terminal residues provide specificity, whereas in the case of the Ra-group only N-terminal residues are important. The binding pattern of the Ra-group member yYFB0 more closely resembles that of the Rb-group, and therefore, yYFB0 may represent an intermediate form. In fact, the sequence of yYFB0 differs significantly from the other Ra-group consensus in that it has a valine instead of a tryptophan at position 30, and therefore, it is located outside the Ra-branch in the tree.
For the interaction of L-group members with APPTPPPLPP (short L-peptide) P-2, P-1, L0, and P1 were identified as key residues (Fig. 5D). In addition, position 2 is essential, as shown by length analyses (data not shown), and preferably proline. This results in the L-group binding motif PPLPp.
To validate the hypothesis that the members of the poly-P-group specifically recognize stretches of proline only, a substitutional analysis of PPPLIPPPPPLPP (part of the long L-peptide) was performed. The specific ligand binding pattern of both WW domains comprised only a stretch of five prolines preceded by a hydrophobic residue (Fig. 5E), yielding the preferred poly-P-group binding motif (p/)PPPPP.
Investigation of the poS/poT-group members with substitution analogs of poS/poT-containing peptides showed only two consecutive residues that are essential for recognition (Fig. 5F), with the pattern (poS/poT)P comprising the specifically recognized motif.
Taken together, the significance of arginine, leucine, phospho-serine/threonine, and proline only as the specificity-determining residues within the ligands for the respective groups was confirmed by these analyses. A general property of all ligand motifs, except for the poS/poT-group, is a pair of two consecutive prolines, which cannot be substituted at all. The position of this pair with respect to the specificity-determining residue differs between the groups. In the case of the Y-group and Ra-group, the two consecutive prolines are separated by one residue from the specificity-determining residue, whereas in the case of the Rb- and L-group this pair directly precedes the specificity-determining residue within the ligand.
The validity of our classification, until now based on the detected recognition profiles and group consensi, is confirmed by the finding of group-specific binding motifs.
Regulation by ligand phosphorylation
It has previously been shown that certain WW domain–ligand interactions may be regulated by ligand phosphorylation (Sudol and Hunter 2000). To investigate this further, a peptide library consisting of 600 potential tyrosine, serine, and threonine kinase target sequences (present in duplicate in phosphorylated and nonphosphorylated form) was screened with the poS/poT-group members hPIN1, yESS1 (Table 1). Both bound a large number of phospho-serine- or phospho-threonine- but no phospho-tyrosine-containing peptides. For hPIN1, selective binding to the phosphorylated peptide only was observed for all 38 binding sequences and dominantly for yESS1 (19 peptides in the phosphorylated form only, seven in both and five in the nonphosphorylated form only). In accordance with the substitutional analyses the common denominator of the identified peptides is (poS/poT)P, with the most strongly binding peptides containing poTPP. Interestingly, phospho-threonine-containing peptides bound, in general, more strongly compared to phospho-serine-containing peptides. Replacing phospho-serine by phospho-threonine in previously identified phospho-serine-containing ligands always increased affinity, substantiating this preference for phospho-threonine (data not shown).
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, fourth result column). For seven WW domains, a chemical shift change was detected, and in three cases strong chemical shift perturbations were observed, although hFBP21-1 and -2 probably interacted with this peptide due to the presence of an arginine. The fact that the NMR screening results for the poT-peptide and the poS-model-peptide did not correlate (only hPIN1 and yYPR152C recognized both) can be attributed to the different positioning of the prolines with respect to the phosphorylated residue.
In addition to these phospho-serine/threonine-recognizing WW domains the NMR screening also showed that 20 of the 42 WW domains recognize the poY-peptide. This raised the question of WW domain selectivity towards peptides phosphorylated on tyrosine, and could imply a WW domain-poY-ligand interaction regulation. Therefore, we complemented the NMR screens by cellulose-bound model peptide arrays of the type GPPPPBG (where B = 19 natural L-amino acids, cysteine omitted, poS, poT, and poY) incubated with selected tyrosine and phospho-tyrosine recognizing domains. hYAP65, yRSP5-3, and mITCHY-1 bound peptides where B was F or Y (Fig. 6A). yRSP5-3 and mITCHY-1 additionally bound the peptide where B was poY, with mITCHY-1 preferring the poY-peptide. Fluorescence titrations of the Y- and poY-peptides with selected WW domains, which showed a chemical shift change larger than 0.15 ppm, yielded dissociation constants below 200 µM (Fig. 6B). This correlation between chemical shift change and dissociation constant supports our approach of estimating binding affinities from chemical shift changes. In general, a weak preference for the nonphosphorylated Y-peptide is observed, with the exception of mITCHY-1 binding with higher affinity to the poY-peptide. It is noteworthy that potential phosphorylation sites are predicted for B1PPB2Y-type motifs (where B1 preferentially equals S,E,D, ... and B2 preferentially equals D,K,E, ...) (Blom et al. 1999) and therefore, mITCHY-1 might be an example of phospho-tyrosine recognition.
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Prediction of WW domain binding activities
Based on our experimental results we attempted to predict tyrosine/phospho-tyrosine recognition propensity for the 482 WW domain sequences found in the SMART database (as of 11/15/2001, sequence redundancy was not removed) (Schultz et al. 1998). To achieve this, a profile HMM (Eddy 1998) based on the sequences of the Y-branch was calculated and used for classifying the WW domain sequences. Two hundred twenty-three WW domains were predicted as Y-branch members, including WW domains of the protein families NEDD4-like ubiquitin ligases (138 of 155 protein family members were classified into the Y-branch), membrane associated guanylate kinases (24 of 36), WW containing oxidoreductases (24 of 24), YAP and homologs (9 of 9), Dystrophin (5 of 21), and PEPP2 (2 of 2). Members of these protein families were already present in our set of 42 WW domains. New candidates for tyrosine/phospho-tyrosine recognition include WW domains of the BAG-family molecular chaperone regulator-3 (hBAG3, ID: O95817; mBAG3, ID: Q9JLV1), the syntaxin4-interacting protein (mSYNIP; ID: Q9WV89) and the zinc finger protein 2 from Trypanosoma brucei (tbZFP2; ID: AAK39107). Solid-phase binding studies with cellulose-bound model peptide arrays of the type GPPPPBG (where B = 19 natural L-amino acids, cysteine omitted, poS, poT, and poY) were performed with these candidates and all bound only peptides where B was F or Y (Fig. 6A). mSYNIP bound additionally GPPPPpoYG but with lower affinity. Substitutional analyses of the ligand GTPPPPYTVG incubated with these candidates revealed the Y-group-specific binding motif PPxY (shown for hBAG3 in Fig. 5A). This altogether validates our prediction for the Y-group.
The exclusive occurrence of all investigated NEDD4like ubiquitin ligase WW domains in the Y-branch (except hSMURF1-1) suggests that binding to tyrosine/phospho-tyrosine-containing peptides is a general feature of this protein family. Moreover, the application of the Y-branch profile HMM to the SMART database WW domain sequences classified the residual NEDD4-like ubiquitin ligases almost exclusively, except for hSMURF1-1 and hSMURF2-2 of various species, in the Y-branch, thus implicating similar binding characteristics. In fact, hSMURF1-1 and hSMURF2-2 and their homologs clearly deviate from the Y-group consensus. This classification by the profile HMM is consistent with in vitro and in vivo studies that indicate that these WW domains interact with proteins containing PY motifs, like ENaC (Kanelis et al. 2001), SMADs (Kavsak et al. 2000), RNA-Polymerase-II (Chang et al. 2000), NF-E2 (Gavva et al. 1997), Atrophin-1 (Wood et al. 1998), and Occludin (Traweger et al. 2002). This can be taken as statistical evidence for the in vivo relevance of this interaction.
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Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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All WW domains were synthesized with a cysteine attached to their N-terminus either to allow for immobilization on a cellulose membrane or for labeling with peroxidase via a Michael-Addition (see sections below, concerning cellulose-bound peptide libraries). To avoid immobilization and labeling at unwanted residues, all cysteines naturally occurring within the WW domain sequences were substituted by serine.
NMR spectroscopy
WW domain NMR samples (200 or 400 µM) were prepared by dissolving 500 µg or 1 mg of lyophylized protein in 500 µL buffer (10 mM phosphate, 100 mM NaCl, 0.1 mM DTT, 0.1 mM EDTA, and 0.02% NaN3 adjusted to a pH of 6.0), followed by the addition of D2O (50 µL). Ligand stock solutions were prepared by dissolving 16 mg of peptide in 400 µL of the same buffer. Ligand was added to the WW domain samples in a twofold molar excess.
All 1D-1H NMR spectra were recorded on a Bruker DRX600 in standard configuration using an inverse triple resonance probe equipped with triple axis self shielded gradient coils. A sample changer able to handle 60 samples was used for the measurements. The spectrometer was operated using the program ICON-NMR on top of XWIN-NMR (Bruker). All experiments were performed at 285 K; water suppression was achieved using a 3–9–19WATERGATE sequence (Sklenar et al. 1993). Five hundred twelve and 1024 scans were taken for samples containing protein concentrations of 400 and 200 µM, respectively. Eight thousand one hundred ninety-two datapoints were recorded for each spectrum. Data were processed and analyzed using XWIN-NMR.
Cellulose-bound peptide libraries
Soluble synthetic WW domains, all N-terminally labeled with cysteine-aminohexanoic acid, were attached via the thiol group to a maleimide-functionalized Whatman 50 cellulose membrane (Whatman) using a spot robot ("WW array"). The membranes were derivatized with two ß-alanines (Kramer et al. 1999). Subsequently, N-maleoyl-ß-alanine (0.6 M in Dimethylacetamide) was activated with N,N‘-Diisopropylcarbodiimide (0.3 M) and coupled to the ß-alanine membrane. The WW domains were then spotted onto the membrane as 1 mM protein solutions (in 0.1 M phosphate buffer, pH 7.4, containing up to 20% Dioxane). The remaining maleimide functions were quenched with 0.3 M ß-Mercaptoethanol in phosphate-buffered saline, pH 7.4.
Arrays of single substitution analogs of ligands ("Substitutional analyses of ligands") and a library containing 600 phosphopeptides ("phosphopeptide library", see below) were generated by semiautomated spot synthesis (Frank 1992; Kramer and Schneider-Mergener 1998) (Abimed; Software LISA, Jerini AG) on Whatman 50 cellulose membranes as described (Kramer et al. 1999).
Phosphopeptide library
In the cellulose-bound phosphopeptide library 600 phosphorylation sites taken from the SWISS-PROT/TrEMBL or PhosphoBase v.2 (Kreegipuu et al. 1999) databases were included. They comprised 360 serine, 121 threonine, and 119 tyrosine phosphorylation sites, each of which was represented by one peptide spot in the library. The peptides had a length of 13 amino acids, with the respective phosphorylation site located at position 8. The whole library was synthesized in duplicate in a phosphorylated and nonphosphorylated form.
Binding studies of cellulose-bound peptides
The membrane-bound libraries (WW array, substitutional analyses of ligands, and phosphopeptide library) were prewashed once with ethanol and three times with Tris-buffered saline (TBS), pH 8.0, and then blocked for 4 h with blocking buffer (blocking reagent [Sigma-Genosys] in TBS containing 5% sucrose). Subsequently, the membranes were incubated with peroxidase-labeled target compounds (peptide ligands [10 µg/mL] or WW domains [50 µg/mL], respectively) in blocking buffer overnight at 4°C and then washed at least four times with TBS. For detection, a chemiluminescence system (Pierce) was applied, and detection was performed using a LumiImagerTM (Boehringer Mannheim GmbH). Peroxidase labeling of the ligands and WW domains was carried out as described (Toepert et al. 2001), linking a maleimide-activated horseradish peroxidase (Pierce) to their N-terminus through a cysteine, which was introduced during peptide synthesis.
Fluorescence spectroscopy
Changes in the fluorescence emission spectra of selected WW domains for varying concentrations of tyrosine- or phospho-tyrosine-containing ligands were used to determine dissociation constants. A 1:1 complex model was assumed for calculation of the dissociation constants. The samples were excited at a wavelength of 298 nm and a slit width of 5 nm. For each titration step the emission spectrum was recorded in the wavelength range of 320–420 nm using a slit width of 10 nm. Binding experiments were performed at 25°C (waterbath controlled) using a Perkin-Elmer Luminescence Spectrometer LS50B. Stock solutions of WW domains (100 µM) and peptide ligands (32 mM) were prepared in 40 mM phosphate buffer with 100 mM NaCl and 50 mM Dithiothreitol, pH 7.2. The final WW domain concentration was 10 µM, and the ligand was added to defined concentrations up to approx. 1 mM.
Tree calculation
The tree was constructed using residues on the ligand-exposed face of the ß-sheet [18, 20, 22–26, 28, 30, 32–35, 37, 39] and W17 only. The WW domain prototype sequence was also included (Macias et al. 2000). Based on sequence distances calculated with PROTDIST using the Dayhoff amino acid exchange model (PAM Matrix) the tree was calculated with NEIGHBOR, using the neighbor joining algorithm (PHYLIP package v3.6a [Felsenstein 1989]). The tree visualization was performed using TREEVIEW (Page 1996).
Profile hidden Markov model
Sequences of the Y-branch were compiled into a profile hidden Markov model calculated with HMMer 2.2 (Eddy 1998). By applying this profile HMM to the set of 42 WW domains, the profile HMM was validated and the classification threshold was determined. Using this threshold, the profile HMM was used to rank the 482 WW domain sequences from the SMART database (as of 11/15/2001, sequence redundancy was not removed) according to their homology with the model.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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