Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain.

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.

Comparative structural and energetic analysis of WW domain-peptide interactions.

WW domains are small globular protein interaction modules found in a wide spectrum of proteins. They recognize their target proteins by binding specifically to short linear peptide motifs that are often proline-rich. To infer the determinants of the ligand binding propensities of WW domains, we analyzed 42 WW domains. We built models of the 3D structures of the WW domains and their peptide complexes by comparative modeling supplemented with experimental data from peptide library screens. The models provide new insights into the orientation and position of the peptide in structures of WW domain-peptide complexes that have not yet been determined experimentally. From a protein interaction property similarity analysis (PIPSA) of the WW domain structures, we show that electrostatic potential is a distinguishing feature of WW domains and we propose a structure-based classification of WW domains that expands the existent ligand-based classification scheme. Application of the comparative molecular field analysis (CoMFA), GRID/GOLPE and comparative binding energy (COMBINE) analysis methods permitted the derivation of quantitative structure-activity relationships (QSARs) that aid in identifying the specificity-determining residues within WW domains and their ligand-recognition motifs. Using these QSARs, a new group-specific sequence feature of WW domains that target arginine-containing peptides was identified. Finally, the QSAR models were applied to the design of a peptide to bind with greater affinity than the known binding peptide sequences of the yRSP5-1 WW domain. The prediction was verified experimentally, providing validation of the QSAR models and demonstrating the possibility of rationally improving peptide affinity for WW domains. The QSAR models may also be applied to the prediction of the specificity of WW domains with uncharacterized ligand-binding properties.

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

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.

Structure of an anti-cholera toxin antibody Fab in complex with an epitope-derived D-peptide: a case of polyspecific recognition.

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence-the only difference is a tyrosine residue in position 7-there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies.

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody.

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T-cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti-cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope-unrelated peptides that specifically bound TE33 with affinities similar to or 100-fold higher than the wild-type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X-ray structure of the wild-type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs.