Binding of a diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: a hydrogen exchange mass spectrometry study.
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.
Catalina
M Isabel
MI
Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
Fischer
Marcel J E
MJ
Dekker
Frank J
FJ
Liskamp
Rob M J
RM
Heck
Albert J R
AJ
D004792:Enzyme Precursors / Q000737:chemistry*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008956:Models, Chemical; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013154:Spleen / Q000201:enzymology*; D000072377:Syk Kinase; D018909:src Homology Domains
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase