Discovery of a Thermophilic Protein Complex Stabilized by Topologically Interlinked Chains

Abstract

A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 °C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6Å crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.

Bacterial CS2 Hydrolases from Acidithiobacillus thiooxidans Strains Are Homologous to the Archaeal Catenane CS2 Hydrolase

“…Catenanes are extremely rare in biology. We are aware of only 3 reports: the gp5 capsid protein of bacteriophage HK97 (63), the bovine mitochondrial peroxiredoxin III (64), and a Pyrobaculum aerophilum citrate synthase in which the catenanes are formed by disulfide bridging between the N-and C-terminal ends of each of the homodimer chains (65). The molecular mechanism of the catenane assembly in the Acidianus A1-3 enzyme is not understood.…”

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

“…Those studies showed structural and biochemical evidence of the thermostabilizing effects of disulfide bonds on several proteins (3,4) and produced genomic and proteomic data that indicated prevailing disulfide bond formation in the cells of such thermophiles. For example, by using two-dimensional diagonal SDS-PAGE, they identified several intermolecular disulfide-bonded proteins from P. aerophilum (5).…”

Structural and functional aspects of the MSP (PsbO) and study of its differences in thermophilic versus mesophilic organisms

“…Many thermostable proteins also have charged residues at their surfaces which can be involved in inter subunit interactions or increase the proteins solubility (Martin et al 2001). An increased number of disulfide bonds and Pro residues relative to mesostable enzymes both increase thermostability by constraining the conformation of the protein and making its unfolded form less favorable (Boutz et al 2007;Vieille and Zeikus 2001 and references there in). Conversely, Gly residues have the most degrees of conformational freedom of any amino acid in the polypeptide back-bone and are expected to encourage flexibility more than other residues.…”