Diversity of polyproline recognition by EVH1 domains

Abstract

Enabled/VASP Homology-1 (EVH1) domains function primarily as interaction modules that link signaling proteins by binding to proline-rich sequences. EVH1 domains are ~115 residues in length and adopt the pleckstrin homology (PH) fold. Four different protein families contain EVH1 domains: Ena/VASP, Homer, WASP and SPRED. Except for the SPRED domains, for which no binding partners are known, EVH1 domains use a conserved hydrophobic cleft to bind a four-residue motif containing 2-4 prolines. Conserved aromatic residues, including an invariant tryptophan, create a wedge-shaped groove on the EVH1 surface that matches the triangular profile of a polyproline type II helix. Hydrophobic residues adjacent to the polyproline motif dock into complementary sites on the EVH1 domain to enhance ligand binding specificity. Pseudosymmetry in the polyproline type II helix allows peptide ligands to bind in either of two N-to-C terminal orientations, depending on interactions between sequences flanking the prolines and the EVH1 domain. EVH1 domains also recognize non-proline motifs, as illustrated by the structure of an EVH1:LIM3 complex and the extended EVH1 ligands of the verprolin family.

Figure 1

The EVH1 fold is a member of the Pleckstrin Homlogy superfamily. Structural comparison of the EVH1 domain from Mena (PDB code: 1EVH) with the PH domain from phospholipase C δ (PDB code: 1MAI) and the phosphotyrosine binding domain (PTB) from tensin-1 (PDB code: 1WVH). The PH fold consists of two perpendicular anti-parallel β-sheets followed by a C-terminal α-helix that assemble to form a ligand binding groove between β-strands 1, 2, 6, and 7. In the PH and PTB domains, this groove is occupied by an additional α-helix that moves the recognition of inositol- (3,4,5) triphosphate and phosphotyrosines peptides by PH and PTB domains, respectively, to distinct regions of the EVH1 domain. Rotation by 90° about the vertical axis (lower panel) shows a side view of the PH fold.

Figure 2

Domain organization and phylogenetic tree of EVH1 domain proteins. A) Protein sequences corresponding to the EVH1 domain boundaries identified by Pfam (95) for human (Homo sapiens, Hs), fly (Drosophila melanogaster, Dm), fish (Danio rerio, Dr), worm (Caenorhabditis elegans, Ce), slime mold (Dictyostelium discoideum, Dd) and yeast (Saccharomyces cerevisiae, Sc) were aligned. NCBI accession numbers for the proteins are indicated in the brackets. Multiple sequence alignments and the phylogenetic analysis were performed using ClustalW version 1.83 (96). EVH1 domains within a particular branch are more related to each other than to those within a particular species. B) Domain organization of representative members from each branch of the four EVH1 protein families. In all families the EVH1 domain is located at the N-terminus and is followed by a variety of domains including proline rich domains (PRD), coiled-coil (CC), low complexity regions (LCR), Ena-VASP homology 2 domains (EVH2), GTPase binding domains (GBD), verprolin-cofilin-acidic motifs (VCA), Sprouty-like cysteine-rich domains (SPR), and c-Kit binding domains (KBD).

Figure 3

Interaction of polyproline containing lignads with their EVH1 domains. A) Ribbon and surface diagrams depicting the interaction of Mena with ActA peptide (PDB code: 1EVH), of Homer-1 with the mGluR peptide (PDB code: 1DDV), of N-WASP with the WIP peptide (PDB code: 2IFS), and SPRED-1 (PDB code: 1XOD). No ligands for SPRED domains are known. The purple surface represents residues that are within 5Å of the bound peptide.

Figure 4

Polyproline recognition by SH3, GYF, WW and EVH1 domains. The interaction of proline rich ligands is shown for the four distinct families that recognize proline rich sequences. A) The SH3 domain from Sem5 (PDB code: 1SEM). B) The GYF domain from CD2BP2 (PDB code: 1L27). C) The WW domain from dystrophin (PDB code: 1EG4). D) The EVH1 domain from N-WASP (PDB code: 2IFS).

Figure 5

EVH1 family members use a conserved aromatic-rich binding site to recognize proline-rich ligands. A) Residues used in the recognition of the proline-rich ligands are conserved in the WASP, Homer, Ena/VASP and SPRED families. However, the orientation of the bound PPII helix is reversed when the Homer and Ena/VASP families are compared with the WASP proteins. B) Representative ligand peptide sequences for the Homer, Ena/VASP and WASP EVH1 domain families.

Figure 6

Atypical EVH1 complexes. A) Three distinct WIP epitopes are required for N-WASP binding. Epitopes correspond to WIP amino acids 454–459 (magenta), 461–468 (gold), and 473–478 (blue). B) Hydrophobic contacts between the aromatic WIP residues Phe 454 and Phe 456 (magenta) and Ala 119 (cyan) from N-WASP. C) The WIP polyproline motif straddles the conserved Trp side chain of the EVH1 domain, but in the opposite orientation relative to peptide ligands for Mena and Homer. D) Lys 477 in WIP epitope 3 makes a conserved salt bridge to N-WASP residue Glu 90. E) Structures of the Mena EVH1 bound to the Tes LIM3 domain (left) and the FPPPP peptide (right) employ overlapping binding sites shown in green on the EVH1 domain surface (center).