A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity - PubMed (original) (raw)

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity

Jamie E Mills et al. J Mol Biol. 2007.

Abstract

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

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Figures

Fig. 1. Distal disulfide bond formation regulates Csk activity

(A) Crystal structure of full-length Csk showing the SH3 and SH2 domains, ATP-binding lobe (A-lobe), peptide-binding lobe (P-lobe), the active-site cleft and the C122-C164 disulfide bond. (B) Time-dependent phosphorylation of poly(Glu4Tyr) by wild-type Csk and Csk-C164A was monitored using 32P kinetic assays. The activity of wild-type Csk was measured in the presence and absence of DTT (1 mM).

Fig. 2. The Csk SH2 domain is in slow exchange between two conformations in solution

**(A)**1H-15N HSQC spectrum of 1.0 mM 15N-labeled Csk SH2 domain. Labeled cross-peaks correspond to residues populating double resonances. Resonances corresponding to two conformers of the same residue are connected by a black line. Boxed regions of the spectrum containing representative double resonances are expanded. (B) 1H-15N HSQC spectrum of the Csk SH2 domain (1.0 mM) in which Cys-164 is mutated to Ala. The same boxed regions of the spectrum are expanded but now show a single resonance for each residue. (C) Oxidation state of the disulfide bond in the SH2 domain specifies conformation. 1D projections of 2D 1H-15N HSQC spectra of the select region representing the dual resonances of G47. HSQC spectra of a 4 mM Csk SH2 sample in the presence of 0 mM, 25 mM, 50 mM, 100 mM and 150 mM DTT were recorded. (D) Heterogeneity maps to a contiguous surface. Residues populating dual resonances in the 1H-15N HSQC spectrum of wild-type Csk SH2 domain are mapped onto the X-ray crystal structure of the Csk SH2 domain and highlighted with blue spheres. Heterogeneity is localized to the region surrounding the disulfide bond, shown with green spheres.

Fig. 3. All atom MD Analysis of the SH2 Domain

(A) Csk SH2 domain colored by change in residue B-factors upon removal of the Cys122-CysS164 disulfide bond. Residues 100–101, 135, 143–144, 148–153 and 156–157 show a positive change in B-factor and are highlighted in red. Residues 93–94, 111–113 and 159–161 show a negative change in B-factor and are highlighted in blue. (B) SH2 domain colored by change in RMSD from active conformation upon removal of disulfide bond. Residues 82, 99–101, 135–136, 143–151, 155, 157–158, 162 and 166 show an increase in RMSD and are shown in red and white indicates no change in the respective parameter upon perturbation.

Fig. 4. Communication between the disulfide bond in the SH2 domain and the active site of Csk

Csk is colored based on changes in strain energy upon removal of Cys122-Cys164 bond. Blue indicates a large decrease in strain, cyan indicates a moderate decrease in strain, red indicates a large increase in strain, and pink indicate a moderate increase in strain. White indicates no significant change in strain energy upon removal of the disulfide bond.

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A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity - PubMed (original) (raw)