HHMI UC Berkeley

Patrick Visperas
Visperas Stamp

Modification by covalent reaction or oxidation of cysteine residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding

Patrick R. Visperas, Jonathan A. Winger, Timothy M. Horton, Neel H. Shah, Diane J. Aum, Alyssa Tao, Tiago Barros, Qingrong Yan, Christopher G. Wilson, Michelle R. Arkin, Arthur Weiss and John Kuriyan

Biochem. J. (2015) 465 (149–161)     (local copy)


Zeta-chain Associated Protein of 70kDa (ZAP-70) and Spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signaling, respectively. They are recruited, via their tandem-SH2 domains, to doubly-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signaling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys 39 in ZAP-70, Cys 206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of hydrogen peroxide, and these two cysteine residues are also necessary for inhibition by hydrogen peroxide. Our findings suggest a mechanism by which reactive oxygen species generated during responses to antigen could attenuate signaling through these kinases, and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.

Figures from the paper

(Click on the small image to get a higher-resolution version.)
Figure 1 from paper

Figure 1 - Structure and domain architecture of ZAP-70 and Syk

(A and C) ZAP-70 (PDB ID: 4K2R) and Syk (PDB ID: 4FL2) consist of two SH2 domains connected by a coiled-coil linker (inter-SH2 linker or interdomain A). This SH2 assembly is connected to the kinase domain by a mostly unstructured linker (SH2-kinase linker or interdomain B). (B and D) Ribbon diagram of ZAP-70 and Syk. Cysteine residues are numbered and C-α carbons of each cysteine residue are shown as red spheres.

Figure 2 from paper

Figure 2 - Covalent compounds and identification of adducts

(A) Chemical structures of compounds A, B and C and adducts A, B and C. Cysteine residues are shown in grey. (B–D) De-convoluted mass spectra of different compounds incubated with ZAP–tSH2. Peaks of 31147 Da and 30569 Da correspond to unlabelled tandem-SH2 module (residues 1–259) and a cleavage product with a C-terminal segment missing (residues 1–253) respectively. Peaks with larger masses correspond to ZAP–tSH2 with mass additions due to covalent modification.

Figure 3 from paper

Figure 3 - Inhibition of ITAM binding to ZAP–tSH2 by thiol-reactive compounds and by H2O2

(A) Compounds A, B and C inhibit ITAM binding in a time-dependent manner. (B) C39S mutation protects ZAP–tSH2 from compound B and C but not A inhibition. (C) ZAP–tSH2 is subject to inhibition by non-reducing conditions. Titrating down reductant increases the rate of inhibition. (E) Addition of H2O2 inhibits 2pY peptide-binding. (D and F) C39S mutation protects from inhibition due to non-reducing conditions as well as by H2O2.

Figure 4 from paper

Figure 4 - Structure of ZAP–tSH2:compound-A

(A) Overall structure of ZAP–tSH2:compound-A shows a novel conformation of the interdomain A inter-SH2 linker. Inset: 2Fo−Fc map shows election density for Cys117–adduct-A. Deformation of the inter-SH2 domain-linker helix-backbone hydrogen bond is compensated for by hydrogen-bond formation between the Asp120 carbonyl and the secondary amine of the guanadino group on Arg124. (B) π-stacking interaction between Cys117–adduct-A and Tyr178 in an adjacent molecule contributes toward inter-SH2 domain-linker coil deformation. (C) Superposition of ZAP–tSH2:compound-A structure with that of ZAP–tSH2:ITAM (PDB ID: 2OQ1) shows bound sulfate ions in the same position as ITAM phosphotyrosine residues.

Figure 5 from paper

Figure 5 - Structure of ZAP–tSH2:ITAM:compound-B<

(A) Structure of ZAP–tSH2:ITAM:compound-B overlaid with that of ZAP–tSH2:ITAM previously determined (PDB ID: 2OQ1, shown in grey). (B) Structure of ZAP–tSH2:ITAM:compound-B. Inset: 2Fo−Fc map shows electron density for adduct B formed on Cys78.

Figure 6 from paper

Figure 6 - Inhibition of ITAM binding to Syk–tSH2 by thiol-reactive compounds and by H2O2

(A and B) Compounds and H2O2 inhibit Syk–tSH2 binding to 2pY peptide. (C and D) C206S mutation protects from compound and H2O2 inhibition. (E) C206A mutant demonstrates a similar lack of inhibition by compounds. WT, wild-type.

Figure 7 from paper

Figure 7 - Locations of cysteine residues critical for inhibition in the SH2 phosphotyrasine-binding pockets of ZAP-70 and Syk

(A and B) Rendering is of an overlay of ZAP-70 N-SH2 (above, blue) and Syk C–SH2 (below, purple) domains shown separately. The specific cysteine residue targets of covalent inhibition for both proteins are in a position that would block phosphotyrosine-binding.

Figure 8 from paper

Figure 8 - Introduction of a cysteine residue into the N-SH2 domain of Syk is sufficient for inhibition by covalent compounds and by H2O2

(A) Engineered Syk with ZAP-70-like cysteine residue configuration regains inhibition by compounds B and C. (B) Engineered Syk–tSH2 is also sensitive to inhibition by H2O2.

Figure 9 from paper

Figure 9 - Inhibition of the ITAM binding to ZAP–SH2 by covalent modification requires compound specificity

Neither NEM nor IAM inhibits ZAP–tHS2:2pY binding.

Figure 10 from paper

Figure 10 - Addition of H2O2 to a FRET-based assay of kinase activity does not affect the rate of ZAP-70 kinase activity

Activity is expressed as a ratio of acceptor fluorescence over donor fluorescence.

Supplemental Material (internal only)