The A-chain of Insulin Contacts the Insert Domain of the Insulin Receptor

Abstract

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of ValA3, which projects within a crevice between the A- and B-chains. Engineered receptor α-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a PapB25-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and α-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (GlyA3 and Ser A3) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of AbaA3 and allo-ThrA3 analogs are reduced at least 10-fold; the activity of ThrA3-DKP- insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (AbaA3) or by altered polarity (allo-ThrA3 and ThrA3). Our results provide evidence that ValA3, a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the α-subunit in a hormone-receptor complex. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.