The Crystal Structure of the Periplasmic Domain of the Type II Secretion System Protein EpsM From Vibrio cholerae: The Simplest Version of the Ferredoxin Fold

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Abstract

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12–15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host.

Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V. cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7 Å resolution. The monomer consists of two αββ-subdomains forming a sandwich of two α-helices and a four-stranded antiparallel β-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.

Introduction

Gram-negative bacteria generally have a set of machineries for extracellular secretion of proteins, including the type II, IIIa and IV secretion systems.1 The general secretion pathway (Gsp), or type II secretion pathway, consists of two distinct steps. First, the sec machinery translocates unfolded proteins with an N-terminal signal peptide through the inner membrane into the periplasm, where folding, oligomerization, and post-translational modifications take place. Second, an assembly of 12–15 proteins of the terminal branch of the general secretion pathway then translocates the folded proteins through the outer membrane.2., 3.

A variety of proteins, e.g. pullulanase (∼117 kDa) or the AB5 pentamers of the cholera toxin and the heat-labile enterotoxin (both ∼86 kDa), is excreted via the type II secretion system with a high species-dependent specificity. The type II secretion system has been reported in many human, animal and plant pathogens of the proteobacteria, mostly clustered in the γ-subdivision including species in the genera Aeromonadaceae, Alteromonadaceae, Legionellae, Enterobacteriaceae, Pseudomonaceae, Vibrionaceae and Xanthomonadales.4

Vibrio cholerae uses its type II secretion system for the export of cholera toxin, the causative agent of the devastating human disease cholera. The type II secretion system of V. cholerae consists of 15 proteins, called EpsA-EpsO. Most of the genes (EpsC to EpsN) cluster with partial overlaps on a single operon on the large chromosome 1.3., 5. The EpsD protein forms the actual pore in the outer membrane, whereas all the other proteins are either associated with or reside in the inner membrane. Pseudopilins EpsG-J6 are thought to be a retractable plug for the pore or a piston, which might actively push substrates through the pore. The signal for gating of the pore or the energy for the active transport might come from the cytosolic “secretory” ATPase EpsE. The structure of a truncated form of EpsE has recently been solved in our laboratory,7 whereas no atomic structure of any other component of any type II secretion system has been published so far.

EpsM8 is a 165 residue bitopic inner membrane protein. A short N-terminal domain (residues 1–23) is predicted to be located in the cytosol, followed by the transmembrane domain (24–43) and the C-terminal periplasmic domain (44–165). There is evidence that EpsM is responsible for the polar localization of the Eps system in V. cholerae.9 EpsE is associated with the inner membrane via EpsL, a bitopic inner membrane protein.10 EpsM binds to EpsL, which leads to mutual stabilization.11., 12. EpsM also appears to enhance the interaction between EpsE and EpsL.13 EpsE, EpsL and EpsM are assumed to form a stable complex.3

While little is known about the function of EpsM in the secretion complex, EpsM homologues are conserved (Figure 1) in all known type II secretion systems. The pairwise sequence identity of EpsM with these homologues is between 19 and 56% and mostly around 25–35%. No sequence homologies to other protein families are obvious when searching the Swissprot and TrEMBL data bases with WU-Blast2.14 The closest homologues of EpsM are GspM proteins from other Vibrionaceae species. The GpsM homologues from Escherichia coli strains 06:H1, K10, K12, and from enterotoxigenic E. coli (ETEC), called YghD or GspM, respectively, are virtually the same, and share 34% sequence identity with EpsM. As only ETEC has maintained the gene cluster gspD-gspK, (the other E. coli strains have deleted most of the gsp-genes) only ETEC is able to secrete the heat-labile enterotoxin via the type II secretion system.15 However, E. coli K12 has a gene cluster similar to the eps cluster containing the yhe, hof and psh genes, which are not expressed under normal conditions,15 and PshM shares 19% sequence identity with EpsM.

The sequence homology within the GspM family (see Figure 1) is distributed quite unevenly along the polypeptide chain: the N-terminal part comprising residues 1–22 shows very little homology, except for the highly conserved Trp12 and Arg18, whereas the predicted transmembrane helix is quite conserved. Pro42 at the C terminus of the transmembrane helix is the only residue that is conserved in all species. After this residue there is a block with a high degree of sequence conservation (Glu45-Ser65) followed by a stretch of residues (Glu66-Pro87) with strikingly little sequence homology and a second more conserved region (Leu88-Leu159).

As EpsL and EpsM are the interfaces between the regulating or energy providing EpsE in the cytoplasm and the rest of the type II machinery, a more detailed knowledge of these proteins is vital for an understanding of the gating or the energy transduction in the Eps system. Given the crucial role EpsM plays, its structure may also form a platform for subsequent structure-based drug design. Since full-length EpsM has so far resisted all our attempts at crystallization, we decided to crystallize its periplasmic soluble domain, which comprises 74% of the total protein. The three-dimensional structure has a very simple topology, which appears to be a novel cyclic permutation of a fold seen in a very large family of proteins.

Section snippets

The tertiary and quaternary structure of the periplasmic domain of EpsM

The periplasmic domain of EpsM was cloned and expressed as two different variants, a short (residues 65–165) and a long construct (residues 44–165), see Figure 1, each as C-terminally His6-tagged proteins. Both constructs yielded very similar crystals. Crystals of the short construct with space group P3221 allowed the structure eventually to be determined with Se-Met MAD-phasing methods; however, solving the structure was hampered by a relatively small amount of methionine residues, all of

Structure of the periplasmic domain of EpsM

The structure of the periplasmic domain of EpsM provides the first atomic resolution view on a protein of the type II secretion machinery of V. cholerae located in the inner membrane. Although a deeper understanding on how the signal from EpsE is transduced through the membrane would require crystal structures of both full-length EpsM alone and in complex with other membrane-bound Eps proteins, a significant amount of information can be extracted from the soluble domain of EpsM.

Both full-length

Cloning

On the basis of the predicted topology, sequence conservation and hydrophobicity of residues of EpsM, two constructs for the soluble periplasmic domain were made (cf. Figure 1). The DNA for the constructs was amplified by PCR from plasmid DNA containing full-length EpsM (a gift from M. Sandkvist) using the following primers:

  • long, sense: CCTGTCATGAGCGAGCGTACCGCCCAAGCTC

  • short, sense: CCTGTCATGAGCGAAAACGCCAACGACATCG

  • antisense: CCGCTCGAGGCCTCCACGCTTCAGTTGC

They were cloned in the pET21d(+) plasmid

Acknowledgements

We gratefully acknowledge the staff of beamline 8.2.2 of the Advanced Light Source for their great support during data collection, Stewart Turley, Claire O'Neal and Douglas Davies, for assistance with data collection, and Francis Athappily for assistance in computing. We acknowledge Jay Painter and Ethan A. Merritt for their support during the final stages of the refinement, and Claudia Roach, Konstantin Korotkov, Mark Robien, Ronald Stenkamp and Maria Sandvkist for stimulating discussions.

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