| Popis: |
The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T6, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{T}}_{3}{\mathrm{R}}_{3}^{{\mathrm{f}}}\end{equation*}\end{document}, and R6. Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of HisB5 in wild-type structures: on the T6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in HisB5 pKa, in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of HisB5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of ArgB5-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T6 hexamers rather than R6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3Å, closely resembles the wild-type T6 hexamer. Whereas ArgB5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving HisB5 in wild-type T-states. The substantial receptor-binding activity of ArgB5-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers. |
