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SecA - the ATP motor in the protein translocation machinery

Introduction

In bacteria, the majority of precursor proteins are transported across and integrated into membranes by the Sec system.2;3

Sec comprises among other components the transmembrane protein conduction pore SecYEG and the peripheral SecA ATPase. SecA acts as the molecular motor of the translocation process. It recognizes the signal sequence of a preprotein and utilizes ATP to mediate protein translocation across the membrane while undergoing major structural rearrangements. SecA interacts with various ligands and components along the export pathway like leader and mature regions of preproteins, the SecYEG pore in the membrane, SecB, a small tetrameric cytosolic chaperone, acidic phospholipids, nucleotides, Mg2+ and Zn2+ and its own mRNA. 4 In an early step of the translocation process preproteins are recognized by help of leader sequences that initiate specific binding to SecA. Both the E. coli and the B. subtilis SecA were found to be homodimers in solution under physiological conditions.5 The oligomeric state of the protein during the translocation reaction is subject of a controversial discussion. In solution, SecA forms a homodimer under physiological conditions. It has been described to be translocation active in a dimeric state 6; 7 while other studies propose the dissociation of the dimer during translocation.8; 9 Several lines of evidence indicate that, depending on salt, temperature, 10 specific lipids and signal peptide analogs 11 SecA exists in a dynamic equilibrium of monomeric, dimeric and higher oligomeric states.

SecA is essential in bacteria and there is no SecA analogue in human or animals. Thus, it represents a potential target for antimicrobial agents. Increasing resistance of pathogenic bacteria against the common antibiotics makes it necessary to develop new agents for the therapy of infectious diseases. For this purpose, the knowledge of the three-dimensional structure of the target protein is a prerequisite. The pathogenic microorganism Enterococcus faecalis causes today the majority of human enterococcal infections and is therefore a suitable target for antimicrobial therapy.12

The structures of the Bacillus subtilis monomer and dimer and the M. tuberculosis SecA dimer have recently been determined (Fig. 1). 1; 5; 13 Our work concentrates on studying the structure of SecA from E. faecalis by electron microscopy and x-ray crystallography. The gene coding for SecA from E. faecalis was cloned and overexpressed in E. coli. 14 In this gene, lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapor-diffusion technique. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.


References

1. Sharma, V., Arockiasamy, A., Ronning, D. R., Savva, C. G., Holzenburg, A., Braunstein, M., Jacobs, W. R., Jr. & Sacchettini, J. C. (2003). Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase. Proc. Natl. Acad. Sci. USA 100, 2243-8.

2. Driessen, A. J., Manting, E. H. & van der Does, C. (2001). The structural basis of protein targeting and translocation in bacteria. Nat. Struct. Biol. 8, 492-8.

3. de Keyzer, J., van der Does, C. & Driessen, A. J. (2003). The bacterial translocase: a dynamic protein channel complex. Cell Mol. Life Sci. 60, 2034-52.

4. Vrontou, E. & Economou, A. (2004). Structure and function of SecA, the preprotein translocase nanomotor. Biochim. Biophys. Acta 1694, 67-80.

5. Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B. & Deisenhofer, J. (2002). Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA. Science 297, 2018-26.

6. Driessen, A. J. (1993). SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer. Biochemistry 32, 13190-7.

7. de Keyzer, J., van der Sluis, E. O., Spelbrink, R. E., Nijstad, N., de Kruijff, B., Nouwen, N., van der Does, C. & Driessen, A. J. (2005). Covalently dimerized SecA is functional in protein translocation. J. Biol. Chem. 280, 35255-60.

8. Duong, F. (2003). Binding, activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase. EMBO J. 22, 4375-84.

9. Or, E., Boyd, D., Gon, S., Beckwith, J. & Rapoport, T. (2005). The bacterial ATPase SecA functions as a monomer in protein translocation. J. Biol. Chem. 280, 9097-105.

10. Woodbury, R. L., Hardy, S. J. & Randall, L. L. (2002). Complex behavior in solution of homodimeric SecA. Protein Sci. 11, 875-82.

11. Benach, J., Chou, Y. T., Fak, J. J., Itkin, A., Nicolae, D. D., Smith, P. C., Wittrock, G., Floyd, D. L., Golsaz, C. M., Gierasch, L. M. & Hunt, J. F. (2003). Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA. J. Biol. Chem. 278, 3628-38.

12. Huycke, M. M., Sahm, D. F. & Gilmore, M. S. (1998). Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg. Infect. Dis. 4, 239-49.

13. Osborne, A. R., Clemons, W. M., Jr. & Rapoport, T. A. (2004). A large conformational change of the translocation ATPase SecA. Proc. Natl. Acad. Sci. USA 101, 10937-42.

14. Meining, W., Scheuring, J., Fischer, M. & Weinkauf, S. (2006). Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis. Acta Cryst. F62, 583-5.