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Substrate twinning activates the signal recognition particle and its receptor

Nature volume 427, pages 215–221 (2004)Cite this article

Abstract

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 Å structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3′OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.

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Figure 1: Sequence alignment of SRP GTPases of known structure, and structure of the FtsY–Ffh heterodimer.
Figure 2: The heterodimerization interface.
Figure 3: Functional analysis of conserved residues and the 3′OH of GTP.
Figure 4: Conformational rearrangements on formation of the FtsY–Ffh complex.
Figure 5: The composite catalytic site with the twinned substrates and essential residues.

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Acknowledgements

We thank C. Reyes for invaluable contributions to the initial FtsY mutant design and structure determination of T. aquaticus FtsY·GMPPNP, and R. Vale, H. Bourne and N. Bradshaw for comments on the manuscript. We acknowledge K. Slep and L. Rice for discussion and advice, and thank J. Holton and G. Meigg for support during data collection at the Advanced Light Source. D.F.S was supported by a Burroughs-Wellcome Fund graduate fellowship. S.S. is supported by a Damon Runyan/Walter Winchell Cancer research fellowship. This work was supported by NIH grants to R.M.S. and P.W. P.W is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California at San Francisco, California, 94143-2240, USA

    Pascal F. Egea, Shu-ou Shan, Johanna Napetschnig, David F. Savage, Peter Walter & Robert M. Stroud

  2. Howard Hughes Medical Institute, San Francisco, California, 94143, USA

    Shu-ou Shan & Peter Walter

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  1. Pascal F. Egea
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Correspondence to Pascal F. Egea or Robert M. Stroud.

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Supplementary information

41586_2004_BFnature02250_MOESM1_ESM.mov

Supplementary Movie 1: The complex between FtsY and Ffh bound with GMPPCP. The FtsY/Ffh heterodimer is shown rotating around the quasi-two fold symmetry axis with the twinned nucleotides and the magnesium ions (as in Fig. 1b). (MOV 1187 kb)

41586_2004_BFnature02250_MOESM2_ESM.mov

Supplementary Movie 2: The twinned nucleotides in the composite active site. The two twinned nucleotides are shown together with the essential catalytic residues ( D135(139), R138(142) and Q144(148)) and the attacking waters (as in Figs. 5a and 5b) rotating around the quasi-two fold symmetry axis (MOV 2314 kb)

41586_2004_BFnature02250_MOESM3_ESM.doc

Supplementary Table : Effect of FtsY mutations on the basal GTPase reaction of FtsY and the reciprocally stimulated GTPase reaction of Ffh with FtsY. (DOC 64 kb)

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Egea, P., Shan, So., Napetschnig, J. et al. Substrate twinning activates the signal recognition particle and its receptor. Nature 427, 215–221 (2004). https://doi.org/10.1038/nature02250

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