Abstract
The interaction of the signal-recognition particle (SRP) with its receptor (SR) mediates co-translational protein targeting to the membrane. SRP and SR interact via their homologous core GTPase domains and N-terminal four-helix bundles (N domains). SRP–SR complex formation is slow unless catalyzed by SRP's essential RNA component. We show that truncation of the first helix of the N domain (helix N1) of both proteins dramatically accelerates their interaction. SRP and SR with helix N1 truncations interact at nearly the RNA-catalyzed rate in the absence of RNA. NMR spectroscopy and analysis of GTPase activity show that helix N1 truncation in SR mimics the conformational switch caused by complex formation. These results demonstrate that the N-terminal helices of SRP and SR are autoinhibitory for complex formation in the absence of SRP RNA, suggesting a mechanism for RNA-mediated coordination of the SRP–SR interaction.
Similar content being viewed by others
References
Egea, P.F., Stroud, R.M. & Walter, P. Targeting proteins to membranes: structure of the signal recognition particle. Curr. Opin. Struct. Biol. 15, 213–220 (2005).
Keenan, R.J., Freymann, D.M., Stroud, R.M. & Walter, P. The signal recognition particle. Annu. Rev. Biochem. 70, 755–775 (2001).
Peluso, P., Shan, S.O., Nock, S., Herschlag, D. & Walter, P. Role of SRP RNA in the GTPase cycles of Ffh and FtsY. Biochemistry 40, 15224–15233 (2001).
Miller, J.D., Wilhelm, H., Gierasch, L., Gilmore, R. & Walter, P. GTP binding and hydrolysis by the signal recognition particle during initiation of protein translocation. Nature 366, 351–354 (1993).
Poritz, M.A. et al. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle. Science 250, 1111–1117 (1990).
Miller, J.D., Bernstein, H.D. & Walter, P. Interaction of E. coli Ffh/4.5S ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor. Nature 367, 657–659 (1994).
Phillips, G.J. & Silhavy, T.J. The E. coli ffh gene is necessary for viability and efficient protein export. Nature 359, 744–746 (1992).
Eitan, A. & Bibi, E. The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY. J. Bacteriol. 186, 2492–2494 (2004).
Bernstein, H.D., Zopf, D., Freymann, D.M. & Walter, P. Functional substitution of the signal recognition particle 54-kDa subunit by its Escherichia coli homolog. Proc. Natl. Acad. Sci. USA 90, 5229–5233 (1993).
Powers, T. & Walter, P. Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor. EMBO J. 16, 4880–4886 (1997).
Montoya, G., Svensson, C., Luirink, J. & Sinning, I. Crystal structure of the NG domain from the signal-recognition particle receptor FtsY. Nature 385, 365–368 (1997).
Freymann, D.M., Keenan, R.J., Stroud, R.M. & Walter, P. Structure of the conserved GTPase domain of the signal recognition particle. Nature 385, 361–364 (1997).
Focia, P.J., Shepotinovskaya, I.V., Seidler, J.A. & Freymann, D.M. Heterodimeric GTPase core of the SRP targeting complex. Science 303, 373–377 (2004).
Egea, P.F. et al. Substrate twinning activates the signal recognition particle and its receptor. Nature 427, 215–221 (2004).
Batey, R.T., Rambo, R.P., Lucast, L., Rha, B. & Doudna, J.A. Crystal structure of the ribonucleoprotein core of the signal recognition particle. Science 287, 1232–1239 (2000).
Keenan, R.J., Freymann, D.M., Walter, P. & Stroud, R.M. Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94, 181–191 (1998).
Zopf, D., Bernstein, H.D., Johnson, A.E. & Walter, P. The methionine-rich domain of the 54 kD protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. EMBO J. 9, 4511–4517 (1990).
de Leeuw, E. et al. Membrane association of FtsY, the E. coli SRP receptor. FEBS Lett. 416, 225–229 (1997).
Angelini, S., Boy, D., Schiltz, E. & Koch, H.G. Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites. J. Cell Biol. 174, 715–724 (2006).
Regalia, M., Rosenblad, M.A. & Samuelsson, T. Prediction of signal recognition particle RNA genes. Nucleic Acids Res. 30, 3368–3377 (2002).
Rosenblad, M.A. & Samuelsson, T. Identification of chloroplast signal recognition particle RNA genes. Plant Cell Physiol. 45, 1633–1639 (2004).
Brown, S. & Fournier, M.J. The 4.5 S RNA gene of Escherichia coli is essential for cell growth. J. Mol. Biol. 178, 533–550 (1984).
Peluso, P. et al. Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor. Science 288, 1640–1643 (2000).
Bradshaw, N. & Walter, P. The signal recognition particle (SRP) RNA links conformational changes in the SRP to protein targeting. Mol. Biol. Cell 18, 2728–2734 (2007).
Reyes, C.L., Rutenber, E., Walter, P. & Stroud, R.M. X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates. PLoS ONE 2, e607 (2007).
Gawronski-Salerno, J. & Freymann, D. M. Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY. J. Struct. Biol. 158, 122–128 (2006).
Focia, P.J., Gawronski-Salerno, J., Coon, J.S., V. & Freymann, D.M. Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site. J. Mol. Biol. 360, 631–643 (2006).
Shepotinovskaya, I.V. & Freymann, D.M. Conformational change of the N domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY. Biochim. Biophys. Acta 1597, 107–114 (2002).
Chandrasekar, S., Chartron, J., Jaru-Ampornpan, P. & Shan, S.O. Structure of the chloroplast signal recognition particle (SRP) receptor: domain arrangement modulates SRP-receptor interaction. J. Mol. Biol. 375, 425–436 (2008).
Jagath, J.R., Rodnina, M.V. & Wintermeyer, W. Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP. J. Mol. Biol. 295, 745–753 (2000).
Schmitz, U. et al. NMR studies of the most conserved RNA domain of the mammalian signal recognition particle (SRP). RNA 2, 1213–1227 (1996).
Shan, S.O. & Walter, P. Molecular crosstalk between the nucleotide specificity determinant of the SRP GTPase and the SRP receptor. Biochemistry 44, 6214–6222 (2005).
Sprangers, R. & Kay, L.E. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445, 618–622 (2007).
Gawronski-Salerno, J., Coon, J.S., V., Focia, P.J. & Freymann, D.M. X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases. Proteins 66, 984–995 (2007).
Gariani, T., Samuelsson, T. & Sauer-Eriksson, A.E. Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY. J. Struct. Biol. 153, 85–96 (2006).
Shan, S.O., Stroud, R.M. & Walter, P. Mechanism of association and reciprocal activation of two GTPases. PLoS Biol. 2, e320 (2004).
Lu, Y. et al. Evidence for a novel GTPase priming step in the SRP protein targeting pathway. EMBO J. 20, 6724–6734 (2001).
Chu, F. et al. Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 101, 16454–16459 (2004).
Parlitz, R. et al. Escherichia coli signal recognition particle receptor FtsY contains an essential and autonomous membrane-binding amphipathic helix. J. Biol. Chem. 282, 32176–32184 (2007).
Bahari, L. et al. Membrane targeting of ribosomes and their release require distinct and separable functions of FtsY. J. Biol. Chem. 282, 32168–32175 (2007).
Gross, J.D., Gelev, V.M. & Wagner, G. A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes. J. Biomol. NMR 25, 235–242 (2003).
Goto, N.K., Gardner, K.H., Mueller, G.A., Willis, R.C. & Kay, L.E. A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J. Biomol. NMR 13, 369–374 (1999).
Acknowledgements
The authors would like to thank C. Guthrie, P. Egea and members of the Walter laboratory for insightful comments and careful reading of the manuscript. This work was supported by funding to P.W. from the US National Institutes of Health and the Howard Hughes Medical Institute. The Jane Coffin Childs Memorial Fund supports S.B.N. N.B. was supported by a predoctoral fellowship from the US National Science Foundation. J.D.G. and S.N.F. were supported by the Sandler Family Foundation for Basic Sciences. S.N.F. was supported in part by the Achievement Awards for College Scientists (ARCS) Foundation.
Author information
Authors and Affiliations
Contributions
S.B.N. and N.B. prepared reagents and performed Ffh–FtsY association and dissociation assays; N.B. carried out GTPase experiments; N.B., S.N.F. and J.D.G. designed, executed and interpreted the NMR experiments; S.B.N. performed partial proteolysis assays; S.B.N., N.B. and P.W. wrote the article.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 (PDF 3875 kb)
Rights and permissions
About this article
Cite this article
Neher, S., Bradshaw, N., Floor, S. et al. SRP RNA controls a conformational switch regulating the SRP–SRP receptor interaction. Nat Struct Mol Biol 15, 916–923 (2008). https://doi.org/10.1038/nsmb.1467
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1467
- Springer Nature America, Inc.
This article is cited by
-
Membrane proteins take center stage in Frankfurt
Nature Chemical Biology (2009)
-
It takes two to tango: regulation of G proteins by dimerization
Nature Reviews Molecular Cell Biology (2009)