PDBsum entry 2hel

Signaling protein, transferase

PDB id

2hel

Contents

			Protein chain

					256 a.a.

			Waters ×51

References listed in PDB file

Key reference

Title

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

Authors

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

Ref.

EMBO J, 2006, 25, 4686-4696. [DOI no: 10.1038/sj.emboj.7601315]

PubMed id

16977320

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.



	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.

PROCHECK

	Headers