The Campylobacter jejuni Porin Trimers Pack into Different Lattice Types when Reconstituted in the Presence of Lipid

Abstract

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high-molecular-masc product (120-140 kDa) was observed under mild conditions of solubilization, a folded monomeric form of 35 kDa was seen when treated at high SDS concentrations and finally, a single band around 45 kDa occurred when the sample was heated to 96°C [Bolla, J. M., Loret, E., Zalewski, M. & Pages, J. M. (1995) J. Bucteriol. 177, 4266-42711. The high-molecular-mass product was reconstituted into two-dimensional crystals in the presence of phospholipids and Mg". The C. jejuni porin required different conditions for successful reconstitution into twodimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a = 8.9 nm, b = 14.9 nm, an oblique lattice with a = 8.9 nm, b = 30.1 nm, y = 98", and a trigonal lattice with a = b = 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain-filled indentations.Keywords: Campylobacter jejuni porin; two-dimensional crystallization.Campylohacter jejuni, a gram-negative, microaerophilic bacterium is a inajor cause of acute enteritis and diarrhea in humans throughout the world (Fauchkre and Rosenau, 1991 ;Walker et al., 1988). As with other gram-negative bacteria, Campylobacter species contain outer membrane proteins which facilitate the diffusion of solutes across the outer membrane. The major outer membrane protein (MOMP) of Campylobacter has been identified by Blaser et al. (1983) and Logan and Trust (1982). The MOMP has been shown to possess the characteristic properties of porins (Huyer et al., 1986). More recently, the stability of this porin has been investigated (Bolla et al., 1995). In addition to the denatured monomeric form, two folded species were identified: a stable monomeric conformation and the native trimer, both containing a high level of P-sheet secondary structure characteristic for bacterial porins (Cowan et a]., 1992; Weiss et al., 1991).Here we present three different crystallographic packing arrangements obtained by reconstituting the purified c. jejuni (strain 85H) porin into two-dimensional crystals in the presence of lipids. Negative stain electron microscopy and digital image processing revealed a highly ordered rectangular lattice with unit cell dimensions a = 8.9 C 0.2 nm, b = 14.9 C 0.2 nm, and an oblique lattice a = 8.9 5 0 . 2 nm, h = 30.1 2 0 . 2 nm, y = 98". In addition, a trigonal lattice was observed that had a unit cell size of a = b = 9.620.5 nm, similar to lattices found in native C. jejuni outer membranes (Amako et al., 1996). Correlation averaging was used to calculate projection maps to a resolution of 2 nm. A trimeric protein with three prominent stain-filled indentations was revealed, suggesting that the C. jejuni MOMP is structurally related to the family of the trimeric bacterial porins. MATERIA...

MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca 2+ Ion Bound at the Constriction Zone

“…Analysis of the contacts between monomers using the PISA server [25] (which assesses the likely stability of multimers) identifies this trimer as the stable unit of MOMP. Gel-filtration data point to a trimeric arrangement consistent with electron microscopy of MOMP reconstituted into lipid bilayers [26]. The N-terminal Thr1 (Gly1 in recombinant MOMP) makes hydrogen bonds to strand 1 of the neighboring monomer; consequently, the three N-terminal helices in the trimer form a triangle.…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…The trimeric structure is unstable and is easily dissociated by SDS even at room temperature (79). The trimer conformation, however, has been confirmed in two-dimensional crystallization studies (12,787). Primary sequence analysis utilizing local hydrophobicity and sequence divergence among isolates suggested the presence of 18, rather than 16, transmembrane ␤-strands (362,782), and comparison with specific channels LamB and ScrY (described below) showed potential conservation of some short signature motifs (362).…”

Sequence Polymorphism, Predicted Secondary Structures, and Surface-Exposed Conformational Epitopes of Campylobacter Major Outer Membrane Protein

“…Based on its biochemical and structural features, MOMP has been characterized as a member of the trimeric bacterial porin superfamily (7,16). Purified MOMP from C. jejuni has pore-forming activity when reconstituted in lipid bilayers (10,51). Similar to other gram-negative bacterial porins, MOMP exhibits heat modifiability and can be identified in three conformational forms including the folded monomer (ϳ35 kDa), the denatured monomer (40 to 48 kDa), and the native trimer (120 to 140 kDa) (16).…”