Binding of a Diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: A hydrogen exchange mass spectrometry study

Abstract

Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding. (J Am Soc Mass Spectrom 2005, 16, 1039 -1051

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…[14] Typical electrospray ionization mass spectra of the deuterated Syk tSH2, either ligand-free or bound to the rigid ligand 2, are shown in Figure 6 A and 6 B. The spectrum of ligand-free Syk tSH2 shows a bimodal charge envelope: the more abundant charge envelope is centered around the 12-fold protonated protein ions, whereas the less intense envelope is localized around the 21+ protein ions (Figure 6 A).…”

“…However, to measure the deuterium uptake in the protein, the noncovalent complex was dissociated in the gas phase by application of a high cone voltage in the source interface, through which we monitored the mass of the released protein as described previously. [14] Figure 5. Eyring plots for Syk tandem SH2 domain interacting with immobilized g-ITAM-peptide (1).…”

“…[32] In a global analysis, this program fits the SPR curves from several protein concentrations simultaneously, returning the kinetic parameters of the binding model. Changes in H/D exchange have been associated with conformational changes, [14,[33][34][35] and this inspired us to examine a two-step model with an initial binding event followed by a structural change of the complex from AB1 to AB2 (model 2, Scheme 1). Together with this, a simple bimolecular model in which the protein-ligand structural change is absent was evaluated (model 1, Scheme 1).…”

“…We have previously probed the changes in Syk tSH2 structural dynamics upon binding to the flexible 1 in different protein segments by H/D exchange, proteolysis, and MALDI-MS. [14] The results clearly showed that a major proportion of the decrease in H/D exchange upon binding could be attributed to the overall inter-SH2 linker region and its stabilization by hydrogen bonding. Figure 7 shows that the decreases in H/D exchange for the flexible and the rigid ligand are similar (the maximum mass deviation errors fall around AE 3 Da).…”

“…[13] A role for the inter-SH2 distance can also be inferred from the flexible structure of the helical coiled coil linker between the two SH2 domains. [13,14] Furthermore, tyrosine 130 in the inter-SH2 linker of Syk tSH2 plays a crucial role in binding to the B cell antigen receptor and receptor activation. [15] In view of the high specificity and affinity of bivalent ligands for tSH2, the development of inhibitors of Syk kinase activation would be a very attractive strategy for the treatment of allergic diseases such as asthma [16][17][18] and of breast cancer.…”