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Disorder-order folding transitions underlie catalysis in the helicase motor of SecA

Nature Structural & Molecular Biology volume 13, pages 594–602 (2006)Cite this article

Abstract

SecA is a helicase-like motor that couples ATP hydrolysis with the translocation of extracytoplasmic protein substrates. As in most helicases, this process is thought to occur through nucleotide-regulated rigid-body movement of the motor domains. NMR, thermodynamic and biochemical data show that SecA uses a novel mechanism wherein conserved regions lining the nucleotide cleft undergo cycles of disorder-order transitions while switching among functional catalytic states. The transitions are regulated by interdomain interactions mediated by crucial 'arginine finger' residues located on helicase motifs. Furthermore, we show that the nucleotide cleft allosterically communicates with the preprotein substrate–binding domain and the regulatory, membrane-inserting C domain, thereby allowing for the coupling of the ATPase cycle to the translocation activity. The intrinsic plasticity and functional disorder-order folding transitions coupled to ligand binding seem to provide a precise control of the catalytic activation process and simple regulation of allosteric mechanisms.

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Figure 1: The helicase motor of E. coli SecA is intrinsically dynamic.
Figure 2: The helicase motor domains interact transiently.
Figure 3: Fast backbone motions of isolated IRA2.
Figure 4: Effect on IRA2 induced by ADP binding to SecAΔC.
Figure 5: Thermodynamic analysis of nucleotide binding to SecAΔC and SecA.
Figure 6: Effect of the D649A mutation on SecA properties.
Figure 7: Allosteric effect of the presence of IRA2 and ADP binding to SecAΔC on PBD.
Figure 8: Model of conformational and dynamic changes in the helicase motor of SecA as a function of the nucleotide state, as suggested by the present NMR, thermodynamic and biochemical data.

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Acknowledgements

We wish to thank L. Gierasch for helpful discussions, F. Jordan for the use of the VP-ITC, Y. Papanikolau and K. Petratos for sharing data before publication, S. Papanikou and B. Pozidis for help with chromatography and R. Monteiro, A. Khan and M. Vougioukalaki for experiments performed during their undergraduate training. Initial experiments were supported by an EU large-scale facility grant to SON-NMR Utrecht, The Netherlands. Research was supported by a Scientist Development Grant from the American Heart Association (to C.G.K.), a Johnson & Johnson Discovery Award (to C.G.K.), a Rutgers Busch grant (to C.G.K.), grants from the EU (RTN1-1999-00149, QLK3-CT-2000-00082 and QLK3-CT-2000-00082; to A.E), Pfizer, Inc. and grants from the Greek General Secretariat of Research (01AKMON46 and PENED03ED623; to A.E).

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Author notes

  1. Dimitra Keramisanou and Nikolaos Biris: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Rutgers University, Newark, 07102, New Jersey, USA

    Dimitra Keramisanou, Nikolaos Biris, Ioannis Gelis & Charalampos G Kalodimos

  2. Institute of Molecular Biology and Biotechnology, FORTH, PO Box 1385, Iraklio, GR-71110, Crete, Greece

    Georgios Sianidis, Spyridoula Karamanou & Anastassios Economou

  3. Department of Biology, University of Crete, PO Box 1527, Iraklio, GR-71110, Crete, Greece

    Anastassios Economou

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Correspondence to Charalampos G Kalodimos.

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Supplementary information

Supplementary Fig. 1

Secondary structural elements of IRA2 in solution (PDF 226 kb)

Supplementary Fig. 2

ITC traces and binding isotherms of nucleotide interaction with SecA and SecAΔC (PDF 320 kb)

Supplementary Fig. 3

Genetic complementation assay for secA and secA-D649A genes (PDF 176 kb)

Supplementary Fig. 4

Thermodynamic analysis of nucleotide interaction with SecA, SecA-R574K and SecAΔC (PDF 204 kb)

Supplementary Fig. 5

Effect of 'arginine fingers' mutation on SecA properties (PDF 201 kb)

Supplementary Fig. 6

1H-15N HSQC spectra of PBD, SecAΔC-ΔIRA2 and effect of ADP binding to SecAΔC on PBD (PDF 402 kb)

Supplementary Methods (PDF 155 kb)

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Keramisanou, D., Biris, N., Gelis, I. et al. Disorder-order folding transitions underlie catalysis in the helicase motor of SecA. Nat Struct Mol Biol 13, 594–602 (2006). https://doi.org/10.1038/nsmb1108

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