Molecular Mechanism of Selective Recruitment of Syk Kinases by the Membrane Antigen-Receptor Complex

Abstract

ZAP-70 and Syk are essential tyrosine kinases in intracellular immunological signaling. Both contain an inhibitory SH2 domain tandem, which assembles onto the catalytic domain. Upon binding to doubly phosphorylated ITAM motifs on activated antigen receptors, the arrangement of the SH2 domains changes. From available structures, this event is not obviously conducive to dissociation of the autoinhibited complex, yet it ultimately translates into kinase activation through a mechanism not yet understood. We present a comprehensive theoretical study of this molecular mechanism, using atomic resolution simulations and free-energy calculations, totaling >10 s of simulation time. Through these, we dissect the microscopic mechanism coupling stepwise ITAM engagement and SH2 tandem structural change and reveal key differences between ZAP-70 and Syk. Importantly, we show that a subtle conformational bias in the inter-SH2 connector causes ITAM to bind preferentially to kinase-dissociated tandems. We thus propose that phosphorylated antigen receptors selectively recruit kinases that are uninhibited and that the resulting population shift in the membrane vicinity sustains signal transduction.Non-receptor Syk tyrosine kinases are an important class among eukaryotic signaling enzymes (1). The family consists of ZAP-70 and Syk, which are mostly found in T-and B-cells, respectively. These kinases help to propagate antigen-recognition signals initiated at the cell membrane. To do so, they first associate with the antigen-receptor complex and then phosphorylate downstream signaling and scaffolding proteins (2). Syk kinases are autoinhibited when isolated; activation follows their engagement of so-called ITAM motifs on the intracellular side of the antigen-receptor complex, through a molecular mechanism that is only partially understood. Consistent with their important cellular role, malfunction of Syk kinases underlies a range of immune system disorders (2, 3).Both ZAP-70 and Syk contain a tandem of SH2 modules, which inhibit the catalytic activity of the enzyme by assembling onto the kinase domain. The SH2 tandem (tSH2) 3 also serves as the docking platform for ITAM motifs. These motifs consist of two repeats of sequence YXX(L/I) connected by a spacer of variable length; doubly phosphorylated ITAM motifs are thus suitable targets for SH2 tandems. Moreover, this dual interaction results in greater affinity, typically in the nanomolar range; that of individual SH2 domains is 2 orders of magnitude weaker (4).Despite the high sequence identity of Syk and ZAP-70 (ϳ60%), they differ noticeably in the thermodynamics of ITAM recognition (5-8). From a functional standpoint, this differentiation is likely related to the fact that Syk is more widely expressed, including in non-immune cells; the diversity of its ligands is therefore necessarily broader (3). However, in view of the similarity of the available tSH2 structures (9, 10), the molecular basis for this differentiation is not immediately clear.The molecular mechanism by which ITAM reco...

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

“…In many cases, such as tyrosine kinases and phosphatases, tandems of modular domains also function as intramolecular regulatory units, assembling onto the catalytic components and allosterically inhibiting their mechanisms (33). The emerging paradigm from studies of the structural dynamics of these complexes is that the modular regulatory units have evolved to adopt multiple, well-defined conformations, consistent with their dual functional roles (34). As for other multidomain proteins, the nature of these conformational states seems to be encoded, at least in part, by seemingly nonstructured linkers between individual modules (35); thus, mutations or posttranslational modifications of these linkers often have a significant effect on activity.…”

From in silico target prediction to multi-target drug design: Current databases, methods and applications

“…This can also be useful for understanding the ligand binding energetics associated with individual amino acid substitutions, which may be of interest in the context of specificity within a closely related family of proteins (Fig. 2, example taken from [157]). In the free energy perturbation (FEP) approach, the free energy change is measured as one ligand is "alchemically" transformed into another.…”

An allosteric hot spot in the tandem-SH2 domain of ZAP-70 regulates T-cell signaling

“…The interaction of doubly-phosphorylated ITAM peptide and tSH2 domain of ZAP-70 has extensively studied, but the structural transition of tSH2 domain from the apo (ITAM-Y2P-ζ1 unbound state) to the holo state (ITAM-Y2P-ζ1 bound state) is poorly understood(6, 11, 18, 30–32, 34, 35) (Figure 1b). We used intrinsic tryptophan fluorescence (shown in Figure S1a and S1b) to monitor the structural transition of an isolated tSH2 domain of ZAP-70 upon binding to doubly-phosphorylated ITAM-ζ1 peptide.…”

“…We begin by analyzing the average root-mean-square deviation (RMSD) that provides a qualitative measure of the protein structure and dynamics during the simulation. The average Cα RMSD value of the tSH2 domain bound to ITAM-Y2P-ζ1 (tSH2- holo ) and ITAM-Y2P-ζ1 unbound (tSH2- apo ) structures are 2.79 ± 0.28 Å and 6.44 ± 0.45Å, respectively (Figure 2b and S3a), suggests that the binding of doubly-phosphorylated ITAM peptide quenches the overall backbone dynamics of the tSH2 domain(21, 35). The structure in which either the N-SH2 (N-SH2 ITAM-YP ) or C-SH2 domain (C-SH2 ITAM-YP ) phosphate-binding pocket is occupied by phosphotyrosine residue of ITAM, deviates from the tSH2- holo structure with an average RMSD of 6.40 ± 0.44 Å and 5.45 ± 0.81Å, respectively.…”

“…Through stochastic fluctuations, the two SH2 domains structurally reorient into geometrically close conformation forming the second phosphate-binding pocket (Figure 1b)(18). Alternatively, biochemical analysis and molecular dynamics simulation suggest that the N-SH2 domain may first bind to phosphotyrosine residue of ITAM peptide with low micromolar affinity followed by cooperative binding of second phosphotyrosine to the C-SH2 domain(11, 30, 32, 35).…”