Abstract
SecA is an essential molecular motor for the translocation of proteins across the membrane via the bacterial Sec secretion system. While the Sec system is found in all cells from archaea to multicellular eukaryotes, the SecA protein is mainly found in bacteria. The mechanism of how the motor protein works on a molecular level is still under dispute but it is well established that SecA binds ATP and uses its hydrolysis for the translocation of substrates. In this work, we addressed the question of which conformational changes the protein might undergo during protein translocation. To this end, we investigated the molecular movements of SecA in the absence or the presence of ATP using single-molecule FRET measurements and in silico normal mode analyses. Our results demonstrate that the pre-protein binding domain of SecA is highly dynamic in the absence of the nucleotide and moves towards the helical wing domain in an ATP-bound state.
Highlights
SecA is an essential molecular motor for the translocation of proteins across the membrane via the bacterial Sec secretion system
To understand the movements of SecA domains relative to each other during the translocation process of substrate proteins, which involves the hydrolysis of adenosine triphosphate (ATP), we measured the distance between the protein binding domain (PBD) and the helical wing domain (HWD)
In a SecA derivative where all 4 cysteine codons had been substituted with serine codons, we constructed a double cysteine mutant with one substitution to cysteine in the PBD (K329C) and the other substitution, P704C, in the HWD
Summary
SecA is an essential molecular motor for the translocation of proteins across the membrane via the bacterial Sec secretion system. We addressed the question of which conformational changes the protein might undergo during protein translocation To this end, we investigated the molecular movements of SecA in the absence or the presence of ATP using single-molecule FRET measurements and in silico normal mode analyses. Both the open conformation of the SecA molecule (Osborne et al.[9], PDB: 1TF2) and the wide open conformation of SecA from B. subtilis (Hunt et al.[8], PDB: 1M74) had been crystallised in the presence of adenosine diphosphate (ADP) (Fig. 1). We analysed the conformational change of SecA using in silico experiments (normal mode analysis (NMA)), which confirms our findings
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