Crystal structures of Halothermothrix orenii α-amylase at saturated salt concentration provide insights into protein stability at very high salt

PROTEOMICS, Invitrogen Bioservices India Pvt Ltd* Institute of Molecular and Cell Biology, Singapore** Griffith University, Brisbane***
○Sivakumar Neelamegam* Nan Li** Patel KC Bharat*** Swaminathan Kunchithapadam*

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 crystal structure of a protein, AmyA, a secretory α-amylase isolated from Halothermothrix orenii, which is both halophilic and thermophilic. The crystal structure was determined at 4.7 M NaCl salt concentration at 1.83 Å resolution. To our knowledge this is the most concentrated salt solution from which a protein has been crystallized. 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. When compared to the AmyA structure at low salt, changes in side chain conformations were observed that impart stability to the protein over a wide range of salt concentrations. AmyA binds to more calcium ions and chloride ion when compared to its low salt structure. The structure at high salt reveals novel calcium and chloride binding sites. 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.