PDBsum entry 2p4r

Ligase

PDB id: 2p4r

Contents

Protein chains

55 a.a. *

16 a.a. *

Ligands

SO4

GOL

Waters ×69

* Residue conservation analysis

PDB id:

2p4r

Name:

Ligase

Title:

Structural basis for a novel interaction between aip4 and beta-pix

Structure:

Rho guanine nucleotide exchange factor 7. Chain: a. Fragment: beta-pix sh3 (10-63). Synonym: pak-interacting exchange factor beta, beta-pix. Engineered: yes. E3 ubiquitin-protein ligase itchy homolog. Chain: t. Fragment: aip4 (209-224). Synonym: itch, atrophin-1-interacting protein 4, aip4, nfe2-

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Gene: arhgef7. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: chemically synthesized. Occurs naturally in humans (homo sapien).

Resolution:

2.00Å R-factor: 0.217 R-free: 0.246

Authors:

K.C.Min

Key ref:

J.M.Janz et al. (2007). A novel interaction between atrophin-interacting protein 4 and beta-p21-activated kinase-interactive exchange factor is mediated by an SH3 domain. J Biol Chem, 282, 28893-28903. PubMed id: 17652093 DOI: 10.1074/jbc.M702678200

Date:

13-Mar-07 Release date: 24-Jul-07

Protein chain

O55043  (ARHG7_RAT) -  Rho guanine nucleotide exchange factor 7 from Rattus norvegicus

Seq: 646 a.a.

Struc: 55 a.a.*

Protein chain

Q96J02  (ITCH_HUMAN) -  E3 ubiquitin-protein ligase Itchy homolog from Homo sapiens

Seq: 903 a.a.

Struc: 16 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 1: Chain A: E.C.?
• Enzyme class 2: Chain T: E.C.2.3.2.26 - HECT-type E3 ubiquitin transferase.
• Reaction: S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E2 ubiquitin-conjugating enzyme]-L-cysteine + N₆- ubiquitinyl-[acceptor protein]-L-lysine

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1074/jbc.M702678200

J Biol Chem 282:28893-28903 (2007)

PubMed id: 17652093

A novel interaction between atrophin-interacting protein 4 and beta-p21-activated kinase-interactive exchange factor is mediated by an SH3 domain.

J.M.Janz, T.P.Sakmar, K.C.Min.

ABSTRACT

Cross-talk between G protein-coupled receptors and receptor tyrosine kinase signaling pathways is crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) with the GTP exchange factor beta-p21-activated kinase-interactive exchange factor (beta PIX). We demonstrate that this interaction is mediated in part by the beta PIX-SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimetry is consistent with a heterotrimeric complex with one AIP4-derived peptide binding to two beta PIX-SH3 domains. We determined the crystal structure of the beta PIX-SH3.AIP4 complex to 2.0-A resolution. In contrast to the calorimetry results, the crystal structure shows a monomeric complex in which AIP4 peptide binds the beta PIX-SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of beta PIX. Taken together, the novel interaction between AIP4 and beta PIX represents a new regulatory node for G protein-coupled receptor and receptor tyrosine kinase signal integration. Our structure of the beta PIX-SH3.AIP4 complex provides important insight into the mechanistic basis for beta PIX scaffolding of signaling components, especially those involved in cross-talk.

Selected figure(s)

Figure 3.
AIP4 binds to βPIX-SH3 as a Class I ligand.A, AIP4 peptide binds to a largely hydrophobic surface of βPIX between the RT (depicted in magenta) and n-src (depicted in cyan) loops. In the left-hand view the SH3 ligand-binding surface is seen from above with the peptide lying on the surface. In the right-hand view, the model has been rotated at on oblique angle to give a better view of the second PPII helix. Electron density for the peptide was seen for residues 209-224, although an Ala was placed for the C-terminal Arg, because the side-chain density was not observed. There are two left-handed PPII helices, from residues 209-215 and from 217-223. The second helix represents the core SH3 ligand with Arg-217 occupying the P[-3] specificity pocket, and Pro-220 and Pro-223 occupying the obligate Pro-preferring P[0] and P[3] pockets. A better view of the first PPII helix is seen in the right-hand view. One Arg residue, Arg-214, lies near a negatively charged surface, Arg-211 appears to form a salt bridge with βPIX-SH3, and again two Pro residues, Pro-212 and Pro-215, are buried in shallow pockets on this surface of βPIX-SH3. In the asymmetric unit the N terminus of the peptide appears to point into empty space, but D shows that this end of the peptide interacts closely with a symmetry mate. The surface is colored to show the electrostatic potential as calculated in GRASP, with positive charge represented in blue and negative charge shaded in red. Underlying the surface is a schematic representation of the SH3 backbone. B, a representative section of the calculated electron 2f[o] - f[c] density map is shown at 1.2 σ in a stereo view. AIP4 peptide residues are represented with yellow carbons, whereas those from βPIX-SH3 are shown in green. Arg-217 of AIP4 is shown in this view in close proximity to Glu-24 of βPIX-SH3. C, the N-terminal region of the AIP4 peptide is shown in gray in this view with the symmetry mates as packed in the crystal. The corresponding βPIX-SH3 is shown as molecular surface also in gray with the protein atoms shown also in stick representation. The various symmetry mates are colored so that the corresponding peptide carbon atoms correspond to the color of the molecular surface. Several of the crystal contacts between βPIX-SH3 domains appear to be mediated by electrostatic interactions, whereas this region of AIP4 peptide appears to lie across a hydrophobic surface of a βPIX-SH3 symmetry mate. D, a schematic representation of ligand binding as generated by LIGPLOT. Hydrogen bonds with three AIP4 backbone carbonyl atoms, one of them water-mediated, are shown in the core SH3 ligand (residues 217-223). In addition, the second PPII helix also has one hydrogen bond with a backbone carbonyl, and Arg-211 forms a charge-stabilized hydrogen bond with βPIX-SH3. The peptide is shown with bonds in purple. βPIX-SH3 residues, which form hydrogen bonds with the peptide, are shown with yellow bonds, whereas Van der Waals interactions are depicted by half circles. There is one water-mediated hydrogen bond shown with a cyan sphere representing the water molecule.

Figure 5.
Proposed schematic arrangement of dimer interface of βPIX with AIP4 peptide. An alignment of the core binding regions of the AIP4 and Cbl-b peptides is shown relative to the shallow hydrophobic binding pockets as sites P[-3] to P[+3] as defined previously in two orientations. Below the peptide sequences are the positions of the residues with respect to the structure reported in this report for the AIP4·βPIX complex and for the Class I orientation for the Cbl-b·βPIX complex. Above the peptide sequences are the same positions in the Class II orientation as described in the Cbl-b·βPIX complex, and the proposed Class II orientation of an additional βPIX-SH3 domain based on the ITC data and an analysis of the sequence for AIP4. The proposed interaction of AIP4 in a class II orientation would result in Thr-222 occupying the P[-1] site, which normally favors the presence of a Pro residue. The core sequences are residues 217-224 for AIP4 and 904-911 for Cbl-b, respectively. Underneath each residue are the respective φ/ψ angles, which show how the PPII helix in the case of Cbl-b and PAK2 are distorted. The pockets that favor the presence of Pro residues are indicated by gray squares drawn around the residues.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 28893-28903) copyright 2007.

Figures were selected by an automated process.