Structural basis of protein stability at poly-extreme: Crystal structure of AmyA at 1.6 A resolution

Abstract

Adaptations of proteins to extreme conditions are very important for the survival of extremophiles and have been the subject of study at the atomic level for many years. However, the adaptations of proteins to poly-extreme condition have not been studied in detail so far. Here we report the first crystal structure of a protein, AmyA, a secretory I?-amylase isolated from Halothermothrix orenii, which is both halophilic and thermophilic. The crystal structure was determined at 0.5 M NaCl and 4.7 NaCl salt concentrations at 1.6 and 1.83 ?? resolution, respectively. We observe surprising structural features, which are likely to be the molecular determinants responsible for the extreme stability of AmyA. AmyA lacks the conserved acidic surface, which is considered essential for protein stability at high salinity. Changes in side chain conformations were observed that impart stability to the protein over a wide range of salt concentrations. AmyA binds to two and five calcium ions at low and high salt conditions, respectively. The structure at high salt reveals novel calcium and chloride binding sites. Sedimentation velocity and CD experiments on AmyA reveal the formation of unique reversible poly-dispersed oligomers that show unusually high thermal stability. These studies provide valuable insight into the structural elements that contribute to the stability of AmyA at both physical and chemical extremes and their functional implications.