Fig. 4: Mapping the PIRB binding site on the HACS1 SH3 domain.
(a) A titration was performed with a soluble fusion protein consisting of 6xHis affinity tag, Flag epitope tag, and murine PIRB ITIM3 sequence fused amino-terminally to the Protein G B1 domain (GB1) within an intervening thrombin cleavage site. (b) Superimposed 1H-15N HSQC spectra of 15N-labeled HACS1 SH3 domain (red) and the HACS1 SH3 domain with a ten-fold excess of a PIRB-GB1 protein (blue). Selected peaks with the largest amide chemical shift perturbations (CSPs) are indicated. (c) A plot of weighted 1H/15N amide CSPs. Light and dark blue shading indicate CSPs that exceed a one and two standard deviation threshold, respectively. (d) Triangles above the HACS1 sequence coincide with CSPs of the previous panel. Triangles below sequence indicate residues that comprise a typical peptide binding cleft on an SH3 domain. (e) CSPs from the PIRB-GB1 titration were mapped onto the NMR structure of the HACS1 SH3 domain following the color scheme from the previous panels. For comparison, a typical binding cleft is shown with the same color scheme as the previous panel.
