Two independent mechanisms down-regulate the intrinsic SecA ATPase activity

Area of Science:

• Molecular biology
• Protein translocation
• Biochemistry

Background:

• SecA protein initiates protein translocation via interaction with ATP, preprotein, and SecYEG components.
• SecA undergoes conformational changes, including membrane insertion and ATP hydrolysis, for protein release and subsequent cycles.
• The C-terminal domain of SecA was previously shown to down-regulate ATPase activity by interacting with the N-terminal domain.

Purpose of the Study:

• To investigate the dual inhibitory mechanisms regulating SecA's intrinsic ATPase activity.
• To elucidate the role of a second ATP-binding domain in ATPase regulation.
• To understand the interplay between different regulatory domains of SecA.

Main Methods:

• Utilized a deregulated SecA mutant to study intrinsic ATPase activity.
• Investigated the effect of domain interactions on ATP hydrolysis rates.
• Quantified ATP hydrolysis in the presence and absence of protein translocation.

Main Results:

• Identified a second ATP-binding domain within the N-terminal domain that down-regulates ATPase activity.
• Demonstrated that this N-terminal regulation is independent of the C-terminal domain-mediated regulation.
• Observed a 50-fold increase in translocation-uncoupled ATP hydrolysis in mutants lacking both inhibitory mechanisms.

Conclusions:

• SecA's intrinsic ATPase activity is controlled by at least two independent inhibitory mechanisms.
• The N-terminal domain possesses a second ATP-binding site that contributes to ATPase regulation.
• Understanding these regulatory mechanisms is crucial for comprehending the dynamics of protein translocation.