PDBsum entry 1u3o

Signaling protein

PDB id: 1u3o

Contents

Protein chain

67 a.a.

References listed in PDB file

Key reference

Title

Regulation of rhogef activity by intramolecular and intermolecular sh3 domain interactions.

Authors

M.R.Schiller, K.Chakrabarti, G.F.King, N.I.Schiller, B.A.Eipper, M.W.Maciejewski.

Ref.

J Biol Chem, 2006, 281, 18774-18786. [DOI no: 10.1074/jbc.M512482200]

PubMed id

16644733

Abstract

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.

Figure 4.
FIGURE 4. Mapping of two potential sites on Kal-SH3 for binding of intramolecular PXXP motifs. A, molecular surface representations of Kal-SH3 showing residues affected upon interaction with various PXXP peptides. The lower surface plots represent an 180° rotation about the z axis of the upper surface plots. Residues showing chemical shifts upon interaction with the PXXP peptide are colored as follows. Background was defined as the maximal chemical shift observed for residues in the N and C termini, which are disorderedand should not change upon ligand binding. Residues colored blue have chemical shift changes of >0.05 ppm for the PLSP peptide and >0.14 ppm for the PKTP peptide and represent site 1. Residues colored green have chemical shift changes of >0.14 ppm for the PKTP peptide and >0.06 ppm for the PLPP peptide and represent site 2. Changes in chemical shifts not colored in this figure are Val^21 and Ser^63 for the PLSP peptide and Glu^67 for the PKTP peptide. The canonical PXXP-binding site mapped in other SH3 structures is shown on the far right (colored magenta) B, alignment of Kalirin and Trio N-terminal SH3 domains from different species (upper) with SH3 domains of defined structure with canonical ligand-binding sites (middle) and non-canonical binding sites (lower). The frog Kalirin sequence is derived from an expressed sequence tag that is missing the N-terminal SH3 domain sequence (dashes). The secondary structure of Kal-SH3 is indicated above the sequences and is color-coded as described in the legend to Fig. 2. Kalirin residues that show chemical shift changes of >0.15 ppm upon binding of the PKTP peptide (330 µM) to the SH3 domain (590 µM) are colored blue for site 1 and green for site 2. Numbering is as for the structural determination of the SH3 domain. Residues colored magenta are ligand-binding residues observed in the structures of Fyn (Protein Data Bank code 1AON), c-Src (code 1NLP), Abl (code 1ABO), -spectrin ( -sp; code 1HD3), Grb2 (N terminus; code 1AZE), c-Crk (N terminus; code 1CKA), Sem5 (C terminus; code 2SEM), GADS (code 1H3H), p67^phox (code 1K4U), Pex13p (code 1NM7), Grb2 (C terminus; code 1GFC), and Vav1 (code 1GCQ).

Figure 7.
FIGURE 7. Model of how intramolecular SH3 domain/ligand interactions and intermolecular Kalirin/Crk binding affect GEF activity. Although our data strongly support two PXXP-binding sites in Kal-SH3, as depicted in this model (with site 1 colored blue and site 2 colored green), we cannot rule out a single PXXP-binding site. The PKTP ligand is indicated by light blue ovals, and the PLPP ligand by green ovals. Crk is colored red. The PLSP ligand is located in spectrin repeat 3 (colored cyan). The Sec14p domain and two N-terminal spectrin repeats in Kal8 are not shown.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 18774-18786) copyright 2006.