Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning INSULIN INSERTS A DETACHABLE ARM BETWEEN RECEPTOR DOMAINS

作者：Xu, Bin; Huang, Kun; Chu, Ying-Chi; Hu, Shi-Quan; Nakagawa, Satoe; Wang, Shuhua; Wang, Run-Ying; Whittaker, Jonathan; Katsoyannis, Panayotis G.; Weiss, Michael A.^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2009, 284(21): 14597-14608.

DOI：10.1074/jbc.M900087200

• DIABETES-ASSOCIATED MUTATIONS
• ALANINE-SCANNING MUTAGENESIS
• PHOTO-CROSS-LINKING
• CHAIN ALPHA-HELIX
• B-CHAIN
• CRYSTAL-STRUCTURE
• BINDING-SITE
• BIOLOGICAL-ACTIVITY
• CHIRAL MUTAGENESIS
• NEGATIVE COOPERATIVITY

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic beta-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain beta-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the alpha-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of a beta-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photo-scanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.

• 出版日期2009-5-22