Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

Background: Chaperonin GroEL(D398A) is known to slow down the ATP hydrolysis. Results: ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants are approximate to 20% and < 0.01% of wild-type GroEL, respectively. Conclusion: Asp-52 plays a critical role for ATP hydrolysis of GroEL. Significance: GroEL(D52A/D398A) forms a stable symmetric GroEL-GroES complex, providing a good model to characterize the complex in detail. The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was approximate to 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of approximate to 150 h (approximate to 6 days), providing a good model to characterize the football-shaped complex.