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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

Authors :
Kazi Fahmida Rahman
Scott A. White
Timothy J. Knowles
Guenter Kramer
Damon Huber
Mohammed Jamshad
Douglas G. Ward
Gareth W. Hughes
Bernd Bukau
Fiyaz Mohammed
Publication Year :
2018
Publisher :
Cold Spring Harbor Laboratory, 2018.

Abstract

SecA is an evolutionarily conserved and essential ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. SecA can recognise proteins that are destined for translocation as they are still being synthesised in order to deliver them to the membrane-bound Sec machinery. However, the mechanism of cotranslational substrate recognition is not well defined. In E. coli, SecA contains a relatively long C-terminal tail (CTT), which consists of a small metal-binding domain (MBD) that is attached to the C-terminus of the catalytic core by a flexible linker (FLD). In this study, we investigated the role of the CTT in the interaction of SecA with the ribosome and nascent polypeptides. Previous work indicates that the CTT is required for interaction with the molecular chaperone SecB. However, phylogenetic analysis suggested that the CTT (and the MBD in particular) has an additional function. Binding experiments indicated that the CTT interacts with 70S ribosomes, and disruption of the entire CTT moderately reduced the affinity of SecA for ribosomes. However, disruption of the MBD alone significantly increased the affinity of SecA for ribosomes and inhibited the interaction of SecA with substrate protein, suggesting that the FLD affects the conformation of SecA. Photocrosslinking and mass spectrometry indicated that the FLD is bound at the site where SecA binds to substrate proteins. Structural analysis by x-ray crystallography and small-angle x-ray scattering (SAXS) provided insight into how the CTT influences the structure of SecA in solution. Finally, site-specific crosslinking experiments indicated that binding to nascent substrate protein affects the conformation of SecA. Taken together, our results suggest that the CTT regulates the ability of SecA to interact with substrate proteins.

Details

Language :
English
Database :
OpenAIRE
Accession number :
edsair.doi.dedup.....9f817bafba73c4d51b798d56f32858bb
Full Text :
https://doi.org/10.1101/389460