PDBsum entry 2hel

Signaling protein, transferase

PDB id: 2hel

Contents

Protein chain

256 a.a. *

Waters ×51

* Residue conservation analysis

PDB id:

2hel

Name:

Signaling protein, transferase

Title:

Crystal structure of a mutant epha4 kinase domain (y742a)

Structure:

Eph receptor a4. Chain: a. Fragment: juxtamembrane and kinase domain. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: epha4. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.35Å R-factor: 0.209 R-free: 0.248

Authors:

L.E.Wybenga-Groot,F.Sicheri,T.Pawson

Key ref:

S.Wiesner et al. (2006). A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases. EMBO J, 25, 4686-4696. PubMed id: 16977320 DOI: 10.1038/sj.emboj.7601315

Date:

21-Jun-06 Release date: 13-Feb-07

Protein chain

Q03137  (EPHA4_MOUSE) -  Ephrin type-A receptor 4 from Mus musculus

Seq: 986 a.a.

Struc: 256 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.7.10.1 - receptor protein-tyrosine kinase.
• Reaction: L-tyrosyl-[protein] + ATP = O-phospho-L-tyrosyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1038/sj.emboj.7601315

EMBO J 25:4686-4696 (2006)

PubMed id: 16977320

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases.

S.Wiesner, L.E.Wybenga-Groot, N.Warner, H.Lin, T.Pawson, J.D.Forman-Kay, F.Sicheri.

ABSTRACT

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.

Selected figure(s)

Figure 2.
Figure 2 Comparison of Eph receptor KD crystal structures. (A) Superposition of active Eph KD structures with auto-inhibited EphB2 structures (PDB ID 1JPA). KDs were aligned using C^ atoms of the C-lobes (left panel) and C^ atoms of the N-lobes (right panel). Spheres represent the ordered boundaries of the KD activation segment. (B) Stereo view of kinked KD helices C. The Eph receptor KDs (colored as in panel A) were superimposed using C^ atoms of helix C. The kink stabilizing side chains of Ser677 and Ser680 in auto-inhibited EphB2 JMS-KD are shown in dark blue. (C) View of the inter-lobe cleft, highlighting the ordered regions of the KD activation segments (colored as in panel A). (D) Superposition of EphB2 JMS-KD with the active Eph KD structures, highlighting the region surrounding Tyr750. Backbone traces are colored as in panel A, with all side chains colored according to their respective backbones. The backbone of a typical activation segment conformation from the active insulin RTK (1IR3) is shown in magenta.

Figure 3.
Figure 3 NMR spectral perturbation study of various activation states of EphB2 kinase. (A) Overlay of a representative region of the ^1H,^15N-HSQC spectra of the auto-inhibited EphB2 JMS-KD fragment (black) and the activated EphB2 KD fragment (blue). (B) Residues experiencing spectral perturbations are mapped onto the structure of the EphB2 JMS-KD as relative peak intensities with a linear gradient from white (I/I[ref] than or equal to 0.4) to blue (I/I[ref]=0). Spheres represent the nitrogen atoms of affected residues. (C) As panel A, but for the EphB2 JMS-KD fragment phosphorylated on residues Y604 and Y610 (red). (D) As panel B, but for the phospho-JMS-KD fragment using a linear gradient from white to red. (E) As panel A, but for the EphB2 Y750A JMS-KD mutant (green). (F) As panel B, but for the EphB2 Y750A JMS-KD mutant using a linear gradient from white to green. In all spectral overlays, residues exhibiting significant spectral perturbations are labeled. JMS residues are underlined, whereas residues in the activation segment are in italics. Dotted lines indicate large chemical shift changes between the phosphorylated and unphosphorylated EphB2 JMS-KD fragment, whereas arrows highlight the positions of peaks appearing around 8.0 p.p.m. in the spectrum of the phosphorylated EphB2 JMS-KD.

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 4686-4696) copyright 2006.

Figures were selected by an automated process.