Structure and regulation of SH2 domain from mouse SH2B1 β

SH2B1 is a member of an adaptor protein family that contains two other proteins (SH2B2 and SH2B3), which regulate signalling pathways initiated by hormones such as insulin and leptin.
As a result of alternative splicing of mRNA, four SH2B1 isoforms have been identified (α 765> δ 724> γ 682 > β 670), that differ in their C-terminus downstream of the SH2 domain. The SH2 domain binds to the phosphorylated tyrosine site of receptor tyrosine kinases (RTKs) and causes kinase activation. Additional to the conserved domains of SH2B1 (DD, PH, and SH2), more than 50% of the protein sequence is intrinsically disordered (unstructured region) including the C-terminal tail. The function of the C-terminal diversity is poorly understood. Mouse SH2B1β isoform tail contains a tyrosine residue (Y649), and we hypothesised that pY649 might bind to the tethered SH2 domain and therefore regulate its activity.

The NMR structure of the SH2 domain was calculated using the semi-automated CYANA (version 3.98.5) software. The structure calculation worked, although it required significant manual intervention, including correction of some the automated chemical shift assignments. A new validation method (ANSURR) was applied to check the reliability of the calculated structures, and it is demonstrated that additional hydrogen bond restraints are required to make the rigidity of the structure match experimental data. Phosphorylation using Fer kinase of a construct that includes the SH2 domain and the downstream 45 residues of the intrinsically disordered tail of β, including Y649, caused problems because unexpectedly, Y624 was phosphorylated more rapidly than Y649 and the protein aggregated. We therefore mutated Y624 to Phe. Y624F SH2c was successfully phosphorylated by Fer kinase. 15N HSQC spectra confirmed a binding between pY649 and the pY pocket of the SH2 domain, and identified a new binding pocket in SH2 domain for residues C-terminal of pY. Surprisingly, the affinity between the phosphorylated C-terminus and SH2 was so weak that it could be outcompeted by phosphate in the buffer. The binding affinity between a phospho-tyrosine peptide derived from the C-terminal tail to the SH2 domain was measured using NMR titration. The revealed low affinity was outcompeted with the preferred JAK2 ligand as shown by HSQC spectra. These results provide some new hypotheses about the function of the C-terminal tail of SH2B1β.