Diabetes-Associated Mutations in Human Insulin:  Crystal Structure and Photo-Cross-Linking Studies of A-Chain Variant InsulinWakayama^,

Abstract

Naturally occurring mutations in insulin associated with diabetes mellitus identify critical determinants of its biological activity. Here, we describe the crystal structure of insulin Wakayama, a clinical variant in which a conserved valine in the A chain (residue A3) is substituted by leucine. The substitution occurs within a crevice adjoining the classical receptor-binding surface and impairs receptor binding by 500-fold, an unusually severe decrement among mutant insulins. To resolve whether such decreased activity is directly or indirectly mediated by the variant side chain, we have determined the crystal structure of Leu(A3)-insulin and investigated the photo-cross-linking properties of an A3 analogue containing p-azidophenylalanine. The structure, characterized in a novel crystal form as an R(6) zinc hexamer at 2.3 A resolution, is essentially identical to that of the wild-type R(6) hexamer. The variant side chain remains buried in a nativelike crevice with small adjustments in surrounding side chains. The corresponding photoactivatable analogue, although of low affinity, exhibits efficient cross-linking to the insulin receptor. The site of photo-cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This domain, unrelated in sequence to the major insulin-binding region in the N-terminal L1 beta-helix, is also contacted by photoactivatable probes at positions A8 and B25. Packing of Val(A3) at this interface may require a conformational change in the B chain to expose the A3-related crevice. The structure of insulin Wakayama thus evokes the reasoning of Sherlock Holmes in "the curious incident of the dog in the night": the apparent absence of structural perturbations (like the dog that did not bark) provides a critical clue to the function of a hidden receptor-binding surface.

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

“…2, C and D). Analogous findings have been described in studies of L-Pap A3 and L-Pap A8 insulin analogs (26,27) and were recently extended to Pap A2 and Pap A4 derivatives (37).…”

“…Such stereospecificity suggested that the pro-D ␣-hydrogen of Gly A1 , unlike its pro-L ␣-hydrogen, adjoins a cavity in the hormone-receptor complex. Substitution of Gly A1 by the photoactivatable residue D-Pap results in specific receptor cross-linking; the site of contact (the C-terminal segment of the ␣-subunit) is similar to those mapped in previous studies of L-Pap B25 derivatives (25,32) and corresponding photoactivatable derivatives at positions A3 and A8 (26,27,59).…”

“…Because of the reduced activity of the photostable precursor, photocross-linking studies were performed at equimolar concentrations of the insulin derivative and receptor (200 nM) more than 2000-fold higher than the wild-type dissociation constant. Such high protein concentrations suggest that despite the likelihood of reduced binding affinity, the irradiated solutions contained predominantly 1:1 specific complexes representative of the native high affinity state (27).…”

“…the distinction between an N-terminal amino group of the two-chain hormone versus the neutral peptide linkage of the single-chain grown factor, might indicate a difference in respective receptor environments. Photocross-linking studies employing para-azido-Phe substitutions (25) suggest that these sites are near the hormone-binding surface (26,27) and so might enable modulation of receptor selectivity.…”

Complementation Analysis Demonstrates That Insulin Cross-links Both α Subunits in a Truncated Insulin Receptor Dimer

“…However, a number of studies have shown that the key determinants required for high affinity insulin binding are located in the ␣ subunit N-terminal L1 domain (aa 1-150) and the C-terminal region (aa 704 -719) (2)(3)(4)(5)(6). In particular, Whittaker et al (7,8) have performed an alanine scanning mutagenesis of human IR ␣ subunit and have shown that alanine mutation of Arg 14 , and Val 715 in the C-terminal region resulted in Ͼ10-fold loss in insulin binding affinity. Taken together, these results suggest that insulin interacts simultaneously with these two regions on the ␣ subunit to achieve high affinity binding.…”