Enhancing the activity of a protein by stereospecific unfolding: conformational life cycle of insulin and its evolutionary origins.
A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional beta-strand mediates both self-assembly (within beta-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (Phe(B24)); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the beta-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of Phe(B24), enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.
Hua
Qing-xin
QX
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Xu
Bin
B
Huang
Kun
K
Hu
Shi-Quan
SQ
Nakagawa
Satoe
S
Jia
Wenhua
W
Wang
Shuhua
S
Whittaker
Jonathan
J
Katsoyannis
Panayotis G
PG
Weiss
Michael A
MA