PDBsum entry 2iyb

Metal-binding

PDB id: 2iyb

Contents

Protein chains

114 a.a. *

64 a.a. *

Metals

_ZN ×9

Waters ×384

* Residue conservation analysis

PDB id:

2iyb

Name:

Metal-binding

Title:

Structure of complex between the 3rd lim domain of tes and the evh1 domain of mena

Structure:

Protein enabled homolog. Chain: a, b, c, d. Fragment: evh1 domain, residues 1-113. Synonym: mena. Engineered: yes. Testin. Chain: e, f, g, h. Fragment: 3rd lim domain, residues 357-421. Synonym: tess, tes.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.35Å R-factor: 0.202 R-free: 0.259

Authors:

D.C.Briggs,N.Q.Mcdonald

Key ref:

B.Boëda et al. (2007). Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding. Mol Cell, 28, 1071-1082. PubMed id: 18158903 DOI: 10.1016/j.molcel.2007.10.033

Date:

14-Jul-06 Release date: 16-Oct-07

Protein chains

Q8N8S7  (ENAH_HUMAN) -  Protein enabled homolog from Homo sapiens

Seq: 591 a.a.

Struc: 114 a.a.

Protein chains

Q9UGI8  (TES_HUMAN) -  Testin from Homo sapiens

Seq: 421 a.a.

Struc: 64 a.a.

DOI no: 10.1016/j.molcel.2007.10.033

Mol Cell 28:1071-1082 (2007)

PubMed id: 18158903

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding.

B.Boëda, D.C.Briggs, T.Higgins, B.K.Garvalov, A.J.Fadden, N.Q.McDonald, M.Way.

ABSTRACT

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.

Selected figure(s)

Figure 1.
Figure 1. The LIM3 Domain of Tes Only Binds Directly to the EVH1 Domain of Mena
(A) Schematic representation of Tes, Mena, and zyxin, drawn to scale. The individual domains used in this study are indicated in black.
(B) Immunoblot analysis of glutathione Sepharose pull-downs reveals that GST-LIM3 resin retains GFP-tagged Mena and EVH1, but not the proline-rich (PP), EVH2 domains of Mena or GFP alone. The input (I) and bound (B) samples with the respective GFP-tagged protein and GST resin control are indicated. The ponceau stain demonstrates that all samples contained equivalent amounts of GST or GST-LIM3 resin.
(C) Coomassie-stained gel reveals that His-LIM3 resin binds directly to the GST-EVH1 domain of Mena, but not to that of VASP and Evl or GST control (top panel). All three EVH1 domains, but not GST, are capable of binding directly to the FPPPP-rich domain of zyxin (residues 61–141) (bottom panel). Five percent of the input (I) and 20% of the bound (B) GST-tagged EVH1 domains and His-tagged proteins were loaded. The protein on resin is indicated on the right of the image with an asterisk.
(D) Table showing the identity and divergence between the EVH1 sequences of Mena, VASP, and Evl.
(E) Alignment of the EVH1 sequences of human Mena, Evl, and VASP. Residues highlighted in red indicate the conserved aromatic residues binding FPPPP ligands. Arrowheads indicate the residue at position 12 and the β3-β4 loop that impede interaction of the EVH1 domain of VASP with Tes (see Figure 5).
(F) Binding isotherms titrating the EVH1 domains from Mena (left), VASP (middle), and Evl (right) with the LIM3 domain of Tes. LIM3 interacts with the EVH1 domain of Mena with an apparent affinity of 3.6 μM. In contrast, no interaction with the EVH1 domain of VASP and Evl was detected.
(G) Binding isotherm titrating the EVH1 domain of Mena with the zyxin FPPPP-rich domain indicates an apparent affinity of 7.4 μM.

Figure 2.
Figure 2. The Molecular Structure of the LIM3:EVH1 Complex
(A) Ribbon representation of the LIM3 (green) and EVH1 (blue) heterodimer. Coordinated zinc atoms are shown in pink. The N and C termini are indicated. Side chains for H365, M409, and M420 involved in the EVH1 interface described in the text are shown together with side chains coordinating the zinc atoms.
(B) Both domains were rotated by 90° in opposite directions to show the surface charge complementarity between the largely acidic LIM3 (red) and basic EVH1 (blue) domains. The position of key residues involved in the interface is indicated.
(C) Panel shows two distinct interaction sites (A and B) between the LIM3 and EVH1 domains shown in magenta and described in the text.
(D) Coomassie-stained gel reveals that sequential alanine substitution of the major contact residues in sites A and B of the LIM3 domain progressively weakens its interaction with the EVH1 domain of Mena. The protein on resin is indicated with an asterisk on the right of the gel.

The above figures are reprinted by permission from Cell Press: Mol Cell (2007, 28, 1071-1082) copyright 2007.

Figures were selected by an automated process.