title: Decoding the cryptic active conformation of a protein by synthetic photoscanning: insulin inserts a detachable arm between receptor domains.
abstract: 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 abeta-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 photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Xu
authors.forename: Kun
authors.initials: K
authors.lastname: Huang
authors.forename: Ying-Chi
authors.initials: YC
authors.lastname: Chu
authors.forename: Shi-Quan
authors.initials: SQ
authors.lastname: Hu
authors.forename: Satoe
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Shuhua
authors.initials: S
authors.lastname: Wang
authors.forename: Run-Ying
authors.initials: RY
authors.lastname: Wang
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.forename: Panayotis G
authors.initials: PG
authors.lastname: Katsoyannis
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss