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Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS

Nature volume 453, pages 124–127 (2008)Cite this article

Abstract

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell1. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus2, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II β-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue3. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.

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Figure 1: Structure of the C-terminal domains of EscU and SpaS.
Figure 3: Auto-cleaving mechanism of EscU.
Figure 2: Type III secretion in EPEC and S. typhimurium.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of all the structures have been deposited in the Protein Data Bank with the accession codes 3BZL, 3BZO, 3BZP, 3BZR, 3BZS, 3BZT, 3C03, 3BZV, 3BZX, 3BZY, 3BZZ, 3C00 and 3C01.

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Acknowledgements

We thank T.S., M.C. and P.I.L. for discussions; the staff at the Advanced Light Source beamline 8.2.2 and SSRL beamline 11-1 for data collection time and assistance; and E. Galyov at the Institute for Animal Health for the SipA, SipB and SipC antibodies. This work was supported by an Izaak Walton Killam Research post-doctoral fellowship, a Michael Smith Foundation for Health Research (MSFHR) post-doctoral fellowship and a Canadian Institutes of Health Research (CIHR) post-doctoral fellowship (all to R.Z.). N.C.J.S. and B.B.F. thank the Howard Hughes International Scholar program and the CIHR for funding. N.C.J.S. also thanks the MSFHR and the Canada Foundation of Innovation for infrastructure funding support. N.C.J.S. is also an MSFHR Senior Scholar and CIHR Investigator. S.M. acknowledges grants from the National Institutes of Allergy and Infectious Diseases (NIAID), U54 AI057141 and 5RO1 AI030479.

Author Contributions R.Z. and M.V. cloned and purified EscU and SpaS. R.Z. crystallized EscU and SpaS. R.Z. solved the structures. H.B.F. isolated the chromosomal SpaS mutants. W.D., H.B.F and H.V.N. performed the biochemical experiments in Fig. 2. B.B.F. and S.I.M. provided resources for the experiments shown in Fig. 2. N.C.J.S. provided resources for all data other than those in Fig. 2. All authors discussed the results and commented on the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, and the Center for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada,

    Raz Zarivach, Marija Vuckovic & Natalie C. J. Strynadka

  2. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada ,

    Wanyin Deng & B. Brett Finlay

  3. Department of Microbiology and Medicine, HSB K-140, Box 357710, Seattle, Washington 98195, USA,

    Heather B. Felise, Hai V. Nguyen & Samuel I. Miller

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  1. Raz Zarivach
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  8. Natalie C. J. Strynadka
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Corresponding author

Correspondence to Natalie C. J. Strynadka.

Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-2 and Supplementary Figures 1-13 with Legends. The Supplementary Tables describe the crystallization and the structure determination statistics. The Supplementary Figures describe additional biochemical data, structural analysis and circular dichroism experiments. (PDF 2401 kb)

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Zarivach, R., Deng, W., Vuckovic, M. et al. Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature 453, 124–127 (2008). https://doi.org/10.1038/nature06832

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