A large conformational change of the translocation ATPase SecA - PubMed (original) (raw)
A large conformational change of the translocation ATPase SecA
Andrew R Osborne et al. Proc Natl Acad Sci U S A. 2004.
Abstract
The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria. We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-A resolution. A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides. We propose that the open form of SecA represents an activated state.
Figures
Fig. 1.
Structure of monomeric B. subtilis SecA. Monomeric B. subtilis SecA is presented as a ribbon diagram. NBF1 is shown in yellow, NBF2 is shown in blue, the PPXD is shown in orange, the HSD is shown in green, and the HWD is shown in cyan. ADP is shown in a ball-and-stick representation. The images were prepared by using
molscript
(40),
raster
3
d
(41), or
spock
(available at
http://mackerel.tamu.edu/spock
).
Fig. 2.
Contacts in different SecA crystal forms. (a) Ribbon diagram of the previously determined B. subtilis SecA structure, which is likely to be the physiological dimer (16). (b) The largest contact in the crystal lattice of monomeric B. subtilis SecA. (c) The proposed M. tuberculosis SecA dimer (17). Residues 757–768 in the HSD and the N terminus of SecA that are important for dimerization of E. coli SecA are shown in red and yellow, respectively.
Fig. 3.
Domain movements in monomeric SecA. Ribbon diagram of monomeric B. subtilis SecA in the open conformation (a) and of a single subunit of dimeric B. subtilis SecA in the closed conformation (b). Color codes are as described for Fig. 1. The first and last helices in the PPXD are represented as cylinders to better visualize the transition between the conformations. The arrows in a indicate the movements that are required to convert the open conformation to the closed conformation. The side chains of residues 232 and 773 are shown in red in stick representation. Corresponding E. coli SecA residue numbers are given in parentheses. These residues were mutated to cysteines in E. coli SecA, and the accessibility of residue 824 to a modification reagent was used to probe the transition from the closed to the open conformation.
Fig. 4.
Potential ligand-binding sites in monomeric B. subtilis SecA. (a) A surface representation of SecA showing the surface grooves 1 and 2 in red and blue, respectively. (b) Surface representation of calmodulin with bound peptide shown in green as an α-carbon trace (25). (c) Surface representation of Hsp70, with the C terminus occupying the peptide-binding groove shown in green as an α-carbon trace (26). (d and e) Surface representations of groove 1, showing the location of pockets H and P, respectively. The surface is rendered transparent, and the underlying peptide backbone is color-coded as described for Fig. 1.
Fig. 5.
E. coli SecA can adopt an open conformation in solution. (a) SecA containing single cysteines at positions 824 or 234, or a mutant lacking cysteines (no cys), were labeled with maleimide fluorescein in the presence of the indicated additions. When nucleotide (0.25 mM) was added, 0.5 mM MgCl2 was also included. The samples were separated by SDS/PAGE and visualized under UV light. In the lane labeled quench+SDS, the quenching reagent, either DTT or glycine, was added before labeling or crosslinking. (b) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, separated by SDS/PAGE, and stained with Coomassie blue. The position of dimer crosslinks is indicated. (c) Modification reactions with SecA containing a cysteine at position 824 were performed as in a in the presence of 1-myristoyl-2-hydroxy-_sn_-glycero-3-[phospho-rac-(1-glycerol)] (MLPG, 0.1 mM); 1,2-diheptanoyl-_sn_-glycero-3-phosphocholine (DHPC, 4.2 mM); dodecyl maltoside (DDM, 0.6 mM); decyl maltoside (DM, 3.6 mM); octyl maltoside (OM, 39 mM); octyl glucoside (OG, 36.4 mM); CYMAL4 (15.2 mM); CYMAL6 (1.12 mM); lauryldimethylamine-_N_-oxide (LDAO, 2 mM); digitonin (1%); or SDS (0.5%). (d) In parallel, samples were crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
Fig. 6.
The ATPase site and mutations affecting SecA function. (a) The Walker A motifs of ADP-bound SecA and ATP-bound PcrA are superimposed. The Walker A and B motifs and motif 6 of monomeric B. subtilis SecA are shown in color, and the equivalent motifs in PcrA are shown in gray (18). Arginine residues in motif 6 are shown in stick representation, and a magnesium ion bound to SecA is shown as a green sphere. The ATP bound to PcrA and the ADP bound to SecA are shown in a stick representation in gray and light blue, respectively. The phosphorus atom of the γ-phosphate of ATP bound to PcrA is shown in red. (b) A view of the SecA ATPase site showing that the Walker B motif (red) is connected to the PPXD via a β-strand (gray). The β-strand (blue) that leads from the PPXD back to NBF1 is connected to the strand between the Walker A (black) and B motifs. NBF2 is omitted for clarity, and the remaining domains are color-coded as described for Fig. 1. The aspartate residue in the Walker B motif and ADP are shown in stick representation. (c) SecA mutations that suppress mutations in SecY channel components are shown in yellow, and PrlD mutations that suppress signal sequence mutations are shown in cyan on a backbone representation of monomeric B. subtilis SecA. Residues identified in both screens are shown in green. ADP is shown as a stick representation.
Similar articles
- Conformational flexibility and peptide interaction of the translocation ATPase SecA.
Zimmer J, Rapoport TA. Zimmer J, et al. J Mol Biol. 2009 Dec 11;394(4):606-12. doi: 10.1016/j.jmb.2009.10.024. Epub 2009 Oct 20. J Mol Biol. 2009. PMID: 19850053 Free PMC article. - Structure of a complex of the ATPase SecA and the protein-translocation channel.
Zimmer J, Nam Y, Rapoport TA. Zimmer J, et al. Nature. 2008 Oct 16;455(7215):936-43. doi: 10.1038/nature07335. Nature. 2008. PMID: 18923516 Free PMC article. - The bacterial ATPase SecA functions as a monomer in protein translocation.
Or E, Boyd D, Gon S, Beckwith J, Rapoport T. Or E, et al. J Biol Chem. 2005 Mar 11;280(10):9097-105. doi: 10.1074/jbc.M413947200. Epub 2004 Dec 23. J Biol Chem. 2005. PMID: 15618215 - Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon.
Rusch SL, Kendall DA. Rusch SL, et al. Biochim Biophys Acta. 2007 Jan;1768(1):5-12. doi: 10.1016/j.bbamem.2006.08.013. Epub 2006 Aug 30. Biochim Biophys Acta. 2007. PMID: 17011510 Free PMC article. Review. - [Molecular mechanisms of SecA-mediated protein translocation viewed from structural studies].
Mori H, Tsukazaki T. Mori H, et al. Tanpakushitsu Kakusan Koso. 2009 May;54(6):685-95. Tanpakushitsu Kakusan Koso. 2009. PMID: 19462754 Review. Japanese. No abstract available.
Cited by
- Structural basis of SecA-mediated protein translocation.
Dong L, Yang S, Chen J, Wu X, Sun D, Song C, Li L. Dong L, et al. Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2208070120. doi: 10.1073/pnas.2208070120. Epub 2023 Jan 4. Proc Natl Acad Sci U S A. 2023. PMID: 36598944 Free PMC article. - Cellular dynamics of the SecA ATPase at the single molecule level.
Seinen AB, Spakman D, van Oijen AM, Driessen AJM. Seinen AB, et al. Sci Rep. 2021 Jan 14;11(1):1433. doi: 10.1038/s41598-021-81081-2. Sci Rep. 2021. PMID: 33446830 Free PMC article. - The Structure of Clostridioides difficile SecA2 ATPase Exposes Regions Responsible for Differential Target Recognition of the SecA1 and SecA2-Dependent Systems.
Lindič N, Loboda J, Usenik A, Vidmar R, Turk D. Lindič N, et al. Int J Mol Sci. 2020 Aug 26;21(17):6153. doi: 10.3390/ijms21176153. Int J Mol Sci. 2020. PMID: 32858965 Free PMC article. - Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.
Elfageih R, Karyolaimos A, Kemp G, de Gier JW, von Heijne G, Kudva R. Elfageih R, et al. Protein Sci. 2020 Oct;29(10):2028-2037. doi: 10.1002/pro.3927. Epub 2020 Aug 24. Protein Sci. 2020. PMID: 32790204 Free PMC article. - Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers.
Sanganna Gari RR, Chattrakun K, Marsh BP, Mao C, Chada N, Randall LL, King GM. Sanganna Gari RR, et al. Sci Adv. 2019 Jun 12;5(6):eaav9404. doi: 10.1126/sciadv.aav9404. eCollection 2019 Jun. Sci Adv. 2019. PMID: 31206019 Free PMC article.
References
- Benach, J. & Hunt, J. F. (2004) Nature 427, 24–26. - PubMed
- Mori, H. & Ito, K. (2001) Trends Microbiol. 9, 494–500. - PubMed
- Economou, A. & Wickner, W. (1994) Cell 78, 835–843. - PubMed
- Ramamurthy, V. & Oliver, D. (1997) J. Biol. Chem. 272, 23239–23246. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources