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Comparative Study
. 2003 Mar;12(3):491-500.
doi: 10.1110/ps.0233203.

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

Livia Otte  1 , Urs Wiedemann, Brigitte Schlegel, José Ricardo Pires, Michael Beyermann, Peter Schmieder, Gerd Krause, Rudolf Volkmer-Engert, Jens Schneider-Mergener, Hartmut Oschkinat
Affiliations
Comparative Study

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

Livia Otte et al. Protein Sci. 2003 Mar.

Abstract

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)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.

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Figures

Figure 1.
Figure 1.
Determination of WW domain ligand recognition propensities by NMR spectroscopy and WW arrays. (A) 1D-1H NMR spectroscopy. The spectra typically contained three signals in the downfield region: two originating from the two key tryptophan side-chain NH signals (W17 and W39) in the folded state, and one originating from both tryptophans of residual unfolded material. The assignment of the two tryptophan signals was based on the assumption that the signal of the buried W17 did not change upon ligand addition (Pires et al. 2001). (B, C) The WW array. Interaction of 42 WW domains with the peroxidase-labeled peptides PPGPPPRGPPPR (B) and APPTPPPLPP (C). PPGPPPRGPPPR bound to yYFB0 (Spot #5), hFE65 (Spot #20), hPQBP1 (Spot #22), hHYP109-1 (Spot #26), hFBP21-1 (Spot #27), and hFBP21-2 (Spot #28). APPTPPPLPP bound to hFBP11-1 (Spot #23) and hFBP11-2 (Spot #24). The complete assignment of WW array spot numbers and corresponding WW domains can be found in column 3 (Nr.*) of Figure 2 ▶.
Figure 2.
Figure 2.
Alignment and classification of WW domain sequences according to ligand recognition propensities. The WW domain sequences are grouped according to the experimental results and the clustering obtained by the tree (Fig. 3 ▶). The consensus sequences were calculated for only those domains exhibiting the group-specific recognition activities. Group-specific residues are indicated in bold type. A priori unfolded WW domains are highlighted in gray. The chemical shift changes of the W39 side-chain NH obtained by 1D-1H NMR spectroscopy are summarized in the first four result columns. Small changes are indicated by pale colors (threshold: 0.03 ppm for Y-, poY-, poT/R-, and 0.01 ppm for poS-peptides), large changes by bright colors (threshold: 0.15 ppm for Y-, poY-, poT/R-, and poS-peptides). "Shift" indicates cases in which a chemical shift change for the side-chain NH signal of W39 was not observed or not observable because of the absence of a tryptophan residue at position 39, but other NH signals changed significantly. For WW domains, which do not have a tryptophan residue at position 39, "none" indicates that no other chemical shift perturbations were observable. The results obtained by at least three independent repetitions of WW array screens were classified as strong signals ( + ), weak signals ( +/− ), and no signals ( − ). Serines replacing cysteines are underlined in the sequences. Residues on the ligand-exposed face of the β-sheet are highlighted by a beige background. ID: accession code; Nr.* position on WW array; Sequence**: sequence numbering according to hYAP65 WW domain (Pires et al. 2001), insertion between residues 24 and 25 indicated as 24b; Y (Y-peptide): GPPPPYG; poY (poY-peptide): GPPPPpoYG; poS (poS-peptide): GPPPPpoSG; poT/R (poT/R-peptide): VLpoTPPDRL; Rs (short R-peptide): PPGPPPRGPPPR; Rl (long R-peptide): PPGPPPRGPPPRPPGPPPRGPPPR; Ls (short L-peptide): APPTPPPLPP; L(I)l (long L-peptide): APPTPPPLPPPLIPPPPPLPP.
Figure 3.
Figure 3.
Mapping of ligand recognition propensities onto a tree of sequence similarity for the 42 WW domains. The tree was constructed from a sequence distance matrix, which was calculated using only the residues on the ligand-exposed face of the β-sheet [18, 20, 22–26, 28, 30, 32–35, 37, 39] and W17. The WW domain prototype sequence was included for comparison (Macias et al. 2000). Unfolded WW domains are highlighted in gray. The tree revealed three branches correlating with distinct recognition activities (Y-, Ra-, and Rb-branches). Result coloring according to Figure 2 ▶.
Figure 4.
Figure 4.
Schematic representations of the WW domain β-sheet indicating group-specific sequence patterns. The sequence consensi obtained for the Y-, Ra-, and Rb-groups are compared to the poS/poT-group consensus (residues on the ligand-exposed face are contained within large squares). Allover conserved binding site residues (Y28, S/T37, and W39) are colored in gray. Residues possibly responsible for ligand specificity are colored according to Figure 2 ▶.
Figure 5.
Figure 5.
Substitutional analyses of ligands incubated with the respective WW domains. Each residue within the ligand was substituted by 19 naturally occurring L-amino acids (cysteine omitted). All spots in the left-most column are identical and represent the wild-type peptide. All other spots are single substitution analogs, with rows defining the sequence position that is substituted and columns defining the amino acid that replaces the wild-type residue. The relative spot intensities correlate qualitatively with the binding affinities (Kramer et al. 1999). (A) GTPPPPYTVG incubated with hBAG3 WW domain. (B) PPGPPPRGPPP incubated with hFE65 WW domain. (C) PPGPPPRGPPP incubated with hFBP21-2 WW domain. (D) APPTPPPLPP incubated with hFBP11-1 WW domain. (E) PPPLIPPPPPLPP incubated with yPRP40-2 WW domain. (F) TRHPPVLpoTPPDQE incubated with hPIN1 WW domain.
Figure 6.
Figure 6.
Analysis of phospho-regulation by ligand array and fluorescence spectroscopy. (A) Interactions of peroxidase-labeled WW domains with 22 variants of the peptide GPPPPBG (where B = 19 natural L-amino acids, cysteine omitted, poS, poT, and poY). (B) Dissociation constants determined by fluorescence spectroscopy titrations. The standard deviation calculated while fitting the 1:1 complex model to the titration series data is given in parentheses after the corresponding dissociation constant.
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