Diabetes-associated mutations in human insulin: crystal structure and photo-cross-linking studies of a-chain variant insulin Wakayama.
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.
Wan
Zhu-li
ZL
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Huang
Kun
K
Xu
Bin
B
Hu
Shi-Quan
SQ
Wang
Shuhua
S
Chu
Ying-Chi
YC
Katsoyannis
Panayotis G
PG
Weiss
Michael A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D003920:Diabetes Mellitus / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003432:Cross-Linking Reagents; D007328:Insulin; C048031:insulin, Leu(A3)-; D011972:Receptor, Insulin