The Function of Ile-X-Ile Motif in the Oligomerization and Chaperone-Like Activity of Small Heat Shock Protein AgsA at Room Temperature.

Small heat shock proteins assemble as large oligomers in vitro and exhibit ATP-independent chaperone activities. Ile-X-Ile motif is essential in both the function and oligomer formation. AgsA of Salmonella enterica serovar Typhimurium has been demonstrated to adopt large oligomeric structure and possess strong chaperone activity. Size exclusion chromatography, non-denaturing pore gradient PAGE, and negatively stain electron microscopic analysis of the various C-terminal truncated mutants were performed to investigate the role of Ile-X-Ile motif in the oligomer assembly of AgsA. By measuring the ability to prevent insulin from aggregating induced by TCEP, the chaperone-like activity of AgsA and the C-terminal truncated mutants at room temperature were determined. We found that the truncated mutants with Ile-X-Ile motif partially or fully deleted lost the ability to form large oligomers. Contrast to wild type AgsA which displayed weak chaperone-like activity, those mutants shown significantly enhanced activities at room temperature. In summary, biochemical experiment, activity assay and electron microscopic analysis suggested that Ile-X-Ile motif is essential in oligomer assembly of AgsA and might take the role of an inhibitor for its chaperone-like activity at room temperature.