Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli

PLoS One. 2014 Feb 14;9(2):e86918. doi: 10.1371/journal.pone.0086918. eCollection 2014.

Abstract

The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Secretion Systems*
  • Chromatography, Gel
  • Crystallography, X-Ray
  • Disulfides / metabolism
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism*
  • Escherichia coli Proteins / ultrastructure
  • Light
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Protein Binding
  • Protein Multimerization
  • Scattering, Radiation
  • Sequence Alignment
  • Structural Homology, Protein
  • Surface Plasmon Resonance
  • Virulence Factors / chemistry*
  • Virulence Factors / metabolism*

Substances

  • Bacterial Secretion Systems
  • Disulfides
  • Escherichia coli Proteins
  • HCP1 protein, E coli
  • Mutant Proteins
  • Virulence Factors

Grant support

Work in C.C. laboratory is supported by the CNRS, AMU and by grants from the Fondation de la Recherche Médicale (FRM DEQ2011-0421282) and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. is supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116). Work in E.C. laboratory is supported by the CNRS, the Aix-Marseille Université and funded by a grant from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.