Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

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Abstract

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 Å resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.

Introduction

Outer membrane protein OmpF is a member of non-specific general diffusion porin family of Gram-negative bacteria, such as Salmonella and Escherichia coli (Nakae, 1976, Nikaido, 1994) whose main function is to facilitate the transport of hydrophilic solutes with molecular mass up to 600 Da across the outer membrane (Jap and Walian, 1990, Nikaido, 1993). Salmonella typhi is an obligatory human pathogen that causes typhoid which continues to be a major health problem in developing countries (Crump et al., 2004). Among the newer generation vaccines against typhoid, S. typhi Ty21a vaccine and Vi polysaccharide have proven to be safe (DeRoeck et al., 2007, Ochiai et al., 2007). The S. typhi Ty21a vaccine is orally administered live attenuated vaccine licensed for use in persons 2 years of age or older but requires 3–4 immunisations to induce long-term (at least 6–7 years) protective immunity in two thirds of the immunised individuals (Levine et al., 1999). Interestingly, it has been found that highly immunogenic live oral Salmonella vaccine would ideally be suited as a carrier of genes that express protective antigens cloned from other antigens (Aggarwal et al., 1990, Formal et al., 1981, Wu et al., 1989) and such hybrid recombinant Salmonella vaccines are expected to invoke protective immunity against both the carrier strains as well as the foreign antigens (Fraillery et al., 2007, Hone et al., 1992). In this context, outer membrane proteins (OMPs) of Salmonella have been shown to elicit a protective immunity (Isibasi et al., 1988, Udhayakumar and Muthukkaruppan, 1987). It has also been shown that the Salmonella antiOmpF and antiOmpC antibodies reached maximum bactericidal titres during the secondary response, antiOmpF antibodies being less immunogenic than antiOmpC antibodies (Secundino et al., 2006). S. typhi porins (OmpC) has been shown to display heterologous epitopes on the cell surface (Puente et al., 1995) which can be exploited as vaccine candidate carrying antigens of other disease causing organisms in their loops, making it possible for a double protective therapy.

In addition to their immunological properties as potent surface antigens, porins also act as entry port for various antibiotics (Nikaido, 2003). The bacteria uses either one of the following mechanism to develop antibiotic resistance using porins: loss/reduction of porins, by expression of other porins not involved in antibiotic translocation and by expression of porins with mutations in the key residues involved in the uptake of antibiotics (Delcour, 2009, Pages et al., 2008). Much of the biophysical and mechanistic studies in determining the pathway of antibiotic translocation through porins have focussed on E. coli OmpF, as its structural and functional properties are well understood (Cowan et al., 1992, Danelon et al., 2006). The influx of antibiotics through porins is not just a passive diffusion but involves interactions with key residues in the porin channel and it has been shown in E. coli OmpF that any mutations in these key residues alter the pore properties in terms of diffusion of antibiotics (Bredin et al., 2002, Hajjar et al., 2010b). Hence the crystal structure of porins from different bacterial sources are pre-requisite to understand the specific atomic details, electrostatic pore potential and favourable channel properties involved in antibiotic translocation. Also, structure of porins from pathogenic species like Salmonella will help in designing specific vaccines and improved antibiotics therapy. However, structure determination of membrane proteins is still a bottleneck due to difficulties in producing large amounts of protein and crystallisation. Refolding of porins from inclusion bodies (IBs) and their structure determination was successful in the case of Rhodopseudomonas blastica porin (Schmid et al., 1996) and OpCA from Neisseria meningitidis (Prince et al., 2001) where the refolded proteins showed structural similarity to their native structures. OmpF from E. coli had been overexpressed and refolded in the presence of detergents (Miedema et al., 2004, Visudtiphole et al., 2005). However, this is the first report of crystallisation and structure determination of in vitro refolded OmpF from a human pathogen.

Here we report the crystal structure of OmpF from S. typhi Ty21a at 2.8 Å resolution (PDB: 3NSG).

Section snippets

Overexpression, purification and crystallisation

Genomic DNA was isolated from the vaccine strain of S. typhi Ty21a (Germanier and Furer, 1975). Primers for ompF gene were designed for the mature S. typhi OmpF (SwissProt Accession: Q56113). PCR amplified product was cloned into NdeI and BamHI sites of pET20b (Novagen). E. coli GJ1158 (Bhandari and Gowrishankar, 1997), a salt-inducible overexpression host, was transformed with ompF/pET20b. Protein was expressed into cytoplasmic inclusion bodies (IBs). Purification, solubilisation and refolding

Structure of S. typhi OmpF

The structure of S. typhi Ty21a rfOmpF obeys the construction principle of other general diffusion porins and each monomer barrel has a 16-stranded anti-parallel β-sheet defining an aqueous channel that spans the outer membrane (Fig. 1). S. typhi and E. coli OmpF share higher percentage of similarity at sequence (57.6% identity) (Fig. 2) and structure with the RMSD of 1 Å (Cα). There are eight short beta hairpin turns (T1–T8) at the periplasmic end of the barrel and eight long loops (L1–L8) at

Conclusions

The structure of S. typhi OmpF reported here is from overexpressed inclusion bodies, refolded in vitro in the presence of a zwitterionic detergent. The significantly different packing observed here suggests the possibility of understanding complexes between OmpF and other biologically relevant molecules that can interact at the porin surface. The variations in the exposed surface loops can be used to design OmpF as a potential multivalent vaccine carrier. The asymmetry seen in the pore and the

Acknowledgments

We acknowledge staff of BM14, ESRF, Grenoble, France, DBT, India and Dr. M. Yogavel, and Dr. G. Jasmita for the data collection. Funding and facilities provided by DBT Programme Support, DBT Nanotechnology project, DBT CoE in Bioinformatics and UGC-SAP at School of Biotechnology, MKU are acknowledged. DB and PDK were recipient of fellowship from UGC and CSIR.

References (73)

  • W. Im et al.

    Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics, Brownian dynamics, and continuum electrodiffusion theory

    J. Mol. Biol.

    (2002)
  • P.D. Kumar et al.

    Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC

    Protein Expr. Purif.

    (2005)
  • M.M. Levine et al.

    Duration of efficacy of Ty21a, attenuated Salmonella typhi live oral vaccine

    Vaccine

    (1999)
  • K.L. Lou et al.

    Structural and functional characterization of OmpF porin mutants selected for larger pore size I. Crystallographic analysis

    J. Biol. Chem.

    (1996)
  • H. Miedema et al.

    Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels

    Biophys. J.

    (2004)
  • T. Nakae

    Outer membrane of Salmonella. Isolation of protein complex that produces transmembrane channels

    J. Biol. Chem.

    (1976)
  • H. Nikaido

    Porins and specific diffusion channels in bacterial outer membranes

    J. Biol. Chem.

    (1994)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode. Methods in enzymology

    Macromol. Crystallogr. A

    (1997)
  • D. Petrey et al.

    GRASP2: visualization, surface properties, and electrostatics of macromolecular structures and sequences

    Methods Enzymol.

    (2003)
  • J.L. Puente et al.

    The Salmonella ompC gene: structure and use as a carrier for heterologous sequences

    Gene

    (1995)
  • N. Saint et al.

    Structural and functional characterization of OmpF porin mutants selected for larger pore size II. Functional characterization

    J. Biol. Chem.

    (1996)
  • T. Schirmer et al.

    Brownian dynamics simulation of ion flow through porin channels

    J. Mol. Biol.

    (1999)
  • B. Schmid et al.

    Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure

    FEBS Lett.

    (1996)
  • M.S. Weiss et al.

    The structure of porin from Rhodobacter capsulatus at 1.8 A resolution

    FEBS Lett.

    (1991)
  • P.D. Adams et al.

    PHENIX: building new software for automated crystallographic structure determination

    Acta Crystallogr. D

    (2002)
  • A. Aggarwal et al.

    Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

    J. Exp. Med.

    (1990)
  • R. Benz et al.

    Ion selectivity of gram-negative bacterial porins

    J. Bacteriol.

    (1985)
  • P. Bhandari et al.

    An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer

    J. Bacteriol.

    (1997)
  • J. Bredin et al.

    Colicins, spermine and cephalosporins: a competitive interaction with the OmpF eyelet

    Biochem. J.

    (2003)
  • J. Bredin et al.

    Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region

    Biochem. J.

    (2002)
  • CCP4, 1994. The CCP4 suite: programs for protein crystallography. Acta. Crystallogr. D 50,...
  • S.W. Cowan et al.

    Crystal structures explain functional properties of two E. coli porins

    Nature

    (1992)
  • J.A. Crump et al.

    The global burden of typhoid fever

    Bull. World Health Organ.

    (2004)
  • DeLano, W.L., 2002. The PyMol Molecular Graphic System. DeLano Scientific, San Carlos,...
  • D. DeRoeck et al.

    Putting typhoid vaccination on the global health agenda

    N. Engl. J. Med.

    (2007)
  • B. Dhakshnamoorthy et al.

    Cation-selective pathway of OmpF porin revealed by anomalous X-ray diffraction

    J. Mol. Biol.

    (2009)
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