Mapping polypeptide interactions of the SecA ATPase during translocation

Area of Science:

• Cellular Biology
• Molecular Biology
• Protein Translocation

Background:

• Protein secretion in bacteria relies on the SecA ATPase and the SecY complex.
• The mechanism by which SecA interacts with diverse polypeptide substrates during translocation is not fully understood.
• Structural data suggest SecA may bind substrates within a 'clamp' domain.

Purpose of the Study:

• To investigate the interaction between SecA and translocating polypeptide segments.
• To test the hypothesis that SecA utilizes a clamp mechanism for substrate binding.
• To elucidate the polypeptide path during SecA-mediated protein translocation.

Main Methods:

• Disulfide bridge cross-linking was employed to analyze protein-polypeptide interactions.
• Cysteines were introduced into a translocation intermediate to facilitate cross-linking.
• One cysteine was designed to cross-link to the SecY channel, another to SecA at various positions.

Main Results:

• Evidence supports the hypothesis that translocating polypeptides are captured within SecA's clamp.
• Polypeptides move through the SecA clamp in an extended conformation.
• The polypeptide path into the SecY channel was defined.

Conclusions:

• SecA binds and moves translocating polypeptides through its clamp structure.
• ATP hydrolysis by SecA likely drives polypeptide movement through the SecY channel.
• This study clarifies the mechanism of SecA-mediated protein translocation.