Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site

Abstract

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)

Fig. 1. Overall structure of OmpT. (A) Ribbon representation of OmpT. The extracellular space is located at the top of the figure, and the periplasmic space is at the bottom. Extracellular loops are labelled L1–L5. The position of the membrane bilayer is delineated by horizontal lines. Aromatic residues that are located at the boundary of the hydrophobic and hydrophilic area on the molecular surface are coloured yellow. The proposed catalytic residues are depicted in red, and the purple-coloured residues show the putative LPS-binding site. (B) Stereo representation of a modelled LPS molecule at the putative binding site. The orientation of the OmpT molecule is rotated 90° along the barrel axis, with respect to (A). The OmpT–LPS model was obtained by superimposing the putative LPS-binding site of OmpT onto the LPS-binding site of FhuA (Ferguson et al., 2000). LPS (from the FhuA structure) is shown by thin grey lines. The putative LPS-binding residues in purple are labelled. This figure, and Figures 5 and  7 were prepared using Bobscript (Esnouf, 1997) and Raster3D (Merritt and Bacon, 1997).

Fig. 2. Topology model of the OmpT β-barrel. Amino acid residues are given in one-letter code. Squares represent residues that are present in the β-strands. Side chains of amino acids that are shaded grey point to the outside of the barrel. Extracellular loops are labelled L1–L5 and periplasmic turns are labelled T1–T4. Every 25th residue is marked with the corresponding residue number. The grey area indicates the approximate position of the outer membrane.

Fig. 3. B-factors of the C_α atoms of the two OmpT molecules in the asymmetric unit, i.e. molecule A (solid line) and B (dashed line). β-strands are shown by bars that are black within the membrane-spanning region and grey outside this region. The positions of loops and turns are marked with L1–L5 and T1–T5, respectively.

Fig. 4. Electrostatic surface potential of a cross-section of the β-barrel (orientation identical to Figure 1B). Negatively charged areas are shown in red, positively charged areas in blue. The positions of the proposed catalytic residues are labelled. This figure was produced by the program GRASP (Nicholls et al., 1991).

Fig. 5. Stereo view of a space-filling representation of the active site groove of OmpT, viewed down the β-barrel axis. All conserved residues in the active site are distinctly coloured and labelled. Conserved residues outside the active site are depicted in dark grey and non-conserved residues in light grey. Conserved serines and threonines in the active site are coloured purple, acidic residues red, histidines blue, a tyrosine brown and all hydrophobic residues green. Residues S99, H101 and D210 are labelled with an asterisk, since they are hidden behind other residues. The three mutated residues S99A, G216K and K217G are labelled in black.

Fig. 6. Sequence alignment of OmpT with other members of the omptin family, i.e. OmpP of E.coli, SopA of S.flexneri, PgtE of S.typhimurium and Pla of Y.pestis. All fully conserved residues are shaded grey. The four proposed catalytic residues are depicted with a black background. The approximate position of the 10 β-strands are indicated by arrows and the extracellular loops are labelled L1–L5.

Fig. 7. Stereo view of the proposed catalytic site. (A) The final 2F_o – F_c electron density map, at 2.6 Å resolution and contoured at 1σ (orientation identical to Figure 1B). Residues are shown as sticks. (B) The catalytic site viewed from the top down the β-barrel axis. Active site residues are depicted by ball-and-sticks. Proposed catalytic residues are coloured dark grey and the other residues that are probably involved in substrate binding are coloured light grey. The proposed proteolytic mechanism, in which the His212–Asp210 couple abstract a proton from a water molecule which then attacks the main chain carbon, is represented schematically.