Contribution of TyrB26 to the Function and Stability of Insulin: STRUCTURE-ACTIVITY RELATIONSHIPS AT A CONSERVED HORMONE-RECEPTOR INTERFACE

Michael A Weiss,Vijay Pandyarajan,Nelson B Phillips,Nischay Rege,Michael C Lawrence,Jonathan Whittaker

doi:10.1074/jbc.m115.708347

Michael A Weiss, Vijay Pandyarajan + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m115.708347

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Abstract

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr(B26), a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu(B26) analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr(B26) is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.

Highlights

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface
Receptor-binding Studies Defined Three Classes of Analogs— 18 insulin analogs containing substitutions at B26 were prepared at small scale (Table 1)
A coarse receptor-binding assay was first undertaken that enabled subgroups of the insulin analogs to be distinguished based on displacement of pre-bound 125I-labeled insulin at a uniform analog concentration of 0.75 nM (Fig. 3A)

Summary

Introduction

Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. A co-crystal structure has been determined at 3.5 Å resolution of a ternary complex between insulin, an L1-CR fragment, and a synthetic ␣CT peptide spanning residues 704 –719 of IR-A [4] In this structure (designated the micro-receptor (␮IR) complex) the C-terminal segment of the insulin B chain is detached from the hormone’s ␣-helical core; such detachment is incompatible with classical structures of insulin (Fig. 1C) but enables insertion of this segment (including TyrB26; red and asterisk in Fig. 2A) between the conserved surfaces of L1 and the ␣CT peptide (Fig. 2, B and C). The latter side chains, despite their broad conservation [17] and efficient IR photo-cross-linking as photo-activatable derivatives [15, 18, 19], appear to contact the ␮IR surface only loosely

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Conclusion