Fig 2: 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.

Fig 3: Signaling pathway outcomes involving PIRB and HACS1.(a) Experimental evidence to date places PIRB in an antagonistic role towards several kinds of positive receptor signals, including those from B cell antigen receptor (BCR). Antagonism is achieved by the binding of SHP1 phosphatase to phosphorylated ITIMs, that in turn, prevent critical phosphorylations on BCR by kinases including Syk. The SHP1-PIRB relationship also antagonizes pathways associated with neuronal plasticity. (b) A similar representation of BCR and PIRB signaling that incorporates the HACS1 relationship presented in this study. If HACS1 can either displace or compete with SHP1 for binding PIRB, Syk may remain activated thereby enabling enhanced BCR signaling and neuronal plasticity. In its adaptor role, HACS1 can bring a different set of regulatory and signaling proteins to PIRB.

Fig 4: Peptide array analysis.A series of 12-mers spanning the entire region of the intracellular region of PIRB were used to identify the binding site for the HACS1 SH3 domain. Detection was performed by chemiluminescent antibody-based detection of a 6xHis tag on a purified SH3 domain probe. Strong signals are denoted in the legend as boldface. A series of alanine and glutamic acid substitutions were also performed (rows D and E). Positive controls are indicated by squares on the blot.

Fig 5: A molecular model of the SHP2 SH2 domain with the ITIM3 region of PIRB.To distinguish the regions of ITIM3 that engage SHP1 and HACS1, the peptide is colored blue and magenta, respectively. One amino acid in the model that is outside the SHP2 and HACS1 recognition region is colored green. The sequence CSR makes contacts with SHP2 SH2 domain in this model and also comprises the central portion of the HACS SH3 domain recognition site.