Analysis of β3 Binding to the c-Src SH3 Domain
Authors: Nygren Patrik , Span Lisa M. , Moore David T. , Cheng Hong , Roder Heinrich , DeGrado William F. , Bennett Joel S. 
Published: October 2019

Abstract 383

An essential component of αIIbβ3-mediated outside-in signaling is activation of the tyrosine kinase c-Src, some of which is constitutively bound via its SH3 domain to the C-terminal Arg759-Gly760-Thr761 (RGT) sequence of the β3 cytoplasmic tail. RGT is quite different from the canonical polyproline sequence recognized by SH3 domains in which a polyproline helix packs against a shallow groove composed of aromatic residues (Tyr93, Tyr95, Tyr139 in c-Src). A specificity pocket located at the end of the groove and composed of residues from the n-Src- and RT-loops affects substrate specificity. Because of the obvious difference between RGT and polyproline sequences, we asked how RGT binds to the c-Src SH3 domain and what implications this has for c-Src regulation by αIIbβ3. Initially, we employed CD spectroscopy and tryptophan (Trp) fluorescence because these techniques are sensitive to changes in the local environment surrounding aromatic residues. However, there were no differences in the CD spectrum of the SH3 domain in absence or presence of the β3 peptide NITYRGT, whereas there was a clear shift in the presence of the core polyproline peptide RPLPPLP. Polyproline binding to Trp in the SH3 specificity pocket also results in a blue shift in Trp fluorescence from 355 nm to 347 nm; however, the fluorescence spectrum was essentially unchanged in the presence of NITYRGT. These experiments suggest that either the interaction of NITYRGT with SH3 is extremely weak and not observed at the concentrations used or occurs outside of the aromatic groove and the specificity pocket. Accordingly, we turned to NMR, a method able to detect weak protein-protein interactions. Two dimensional 1H-15N HSQC spectra of the SH3 domain in the presence of NITYRGT exhibited a number of changes in chemical shift compared to the spectrum in the absence of ligand. Sixteen residues located in the n-Src and RT-loops, grouped around the specificity pocket, had chemical shift changes > 0.05 ppm. The largest changes occurred in residues in or adjacent to the RT-loop, especially residues Arg98, Glu100, and Asp102. Of the resides forming the aromatic groove, only Tyr95 which is adjacent to the specificity pocket was perturbed by NITYRGT. Plots of the chemical shift changes for NH groups in SH3 vs. NITYRGT concentration were linear, indicating that the majority of SH3 domain was unbound. Further, a Kd for NITYRGT binding to SH3, estimated from these experiments, was between 175–350 mM. Next, we obtained HSQC spectra for SH3 in the presence of either RPLPPLP or a negative control peptide NITYEGK. Major perturbations due to RPLPPLP occurred in three regions: residues 98–103 (RT-loop), 116–122 (n-Src loop and specificity pocket), and residues 134–138; residues in the aromatic cluster were unaffected by the ligand. By contrast, only a handful of residues showed small perturbations in the presence of NITYEGK and there was no overlap between the affected residues and those affected by RPLPPLP. In conclusion, our results indicate that compared to polyproline sequences, the C-terminus of the β3 cytoplasmic tail binds to the c-Src SH3 domain in the region of the SH3 specificity pocket. Because chemical shifts for acidic residues located in the RT-loop were particularly sensitive to the presence of NITYRGT, it is likely that Arg759 in β3 makes an important contribution to the interaction. Moreover, we found that the interaction between NITYRGT and the c-Src SH3 domain is substantially weaker than was previously reported for the interaction of β3 with c-Src. This suggest the possibility that a third component is required for this interaction to occur under biological conditions. Recently we found that the β3 cytoplasmic tail in solution has weak affinity for the talin-1 FERM domain, but appending the tail to acidic phospholipids increased its affinity by three orders of magnitude. Since the c-Src SH3 domain contains a conserved patch of basic residues that are necessary for binding to acidic phospholipids, it is possible that the interaction of c-Src with β3 is also a ternary interaction in which protein-lipid interactions play an important role.

Disclosures:

No relevant conflicts of interest to declare.