MINI-PROINSULIN AND MINI-IGF-I - HOMOLOGOUS PROTEIN SEQUENCES ENCODING NONHOMOLOGOUS STRUCTURES

Protein minimization highlights essential determinants of structure an d function. Minimal models of proinsulin and insulin-like growth facto r I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wid e interest due to their native foldability but complete absence of bio logical activity. The crystal structure of mini-proinsulin, determined as a T3R3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsuli n under physiologic conditions and compare this structure to that of t he corresponding two-chain analogue. The two proteins each contain sub stitutions in the B-chain (His(B10) --> Asp and Pro(B28) --> Asp) desi gned to destabilize self-association by electrostatic repulsion; the p roteins differ by the presence or absence of a peptide bond between Ly s(B29) and Gly(A1) The structures are essentially identical, resemblin g in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 alpha-hel ix (variable among crystal structures) is less well-ordered in mini-pr oinsulin than in the two-chain variant. The single-chain analogue is n ot completely inactive: its affinity for the insulin receptor is 1500- fold lower than that of the two-chain analogue. Moreover, at saturatin g concentrations mini-proinsulin retains the ability to stimulate lipo genesis in adipocytes (native biological potency). These results sugge st that a change in the conformation of insulin, as tethered by the B2 9-A1 peptide bond, optimizes affinity but is not integral to the mecha nism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms dev iation 4.5 Angstrom) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 A ngstrom). Three-dimensional profile scores suggest that the two struct ures each provide acceptable templates for threading of insulin-like s equences. Mini-proinsulin and mini-IGF-I thus provide examples of homo logous protein sequences encoding non-homologous structures. (C) 1998 Academic Press Limited.