RT Journal Article
SR Electronic
T1 The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 389460
DO 10.1101/389460
A1 Mohammed Jamshad
A1 Timothy J. Knowles
A1 Scott A. White
A1 Douglas G. Ward
A1 Fiyaz Mohammed
A1 Kazi Rahman
A1 Gareth W. Hughes
A1 Günter Kramer
A1 Bernd Bukau
A1 Damon Huber
YR 2018
UL http://biorxiv.org/content/early/2018/08/10/389460.abstract
AB 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.