Structural studies of two outer membrane proteins: OmpT from Escherichia coli and NspA from Neisseria meningitidis (2003)

This Thesis describes the three-dimensional structures of two outer membrane proteins (OMPs), OmpT and NspA, from two pathogenic Gram-negative bacteria. These structures reveal information about the functioning of these proteins and can potentially be used for the design of antimicrobial drugs or vaccines, since they are exposed to the outside of the bacterium. &#13;\nOmpT is present in Escherichia coli, a bacterium that is commensally present in the intestines, and which is the main cause of urinary tract disease. Homologues of OmpT, called omptins, are found in for example Yersinia pestis and Salmonella typhimurium. Omptins are proteases that can cleave extracellular proteins between two consecutive basic amino acids. They are implicated in pathogenicity of several bacteria, since they can cleave plaminogen, which results in fibrinolysis and subsequently the spread of the bacteria. Furthermore, OmpT inactivates defensins, antimicrobial compounds excreted by the epithelial cells of the urinary tract. OmpT had been classified as a serine protease (involving a Ser-His-Asp triad). This classification is, however, controversial. Indeed, site directed mutagenesis studies showed that mutation of Ser99 and His212 led to a significant reduction of catalytic activity. In contrast, commonly used serine protease inhibitors fail to inhibit OmpT activity. Furthermore, OmpT's activity dependent is on the presence of lipopolysaccharide (LPS), a molecule in the outer membrane. To learn more about OmpT's catalytic mechanism and about its LPS dependence, we solved the crystal structure of OmpT (described in chapter 2). The structure reveals a very long ten-stranded b-barrel, with the putative active site in a large cleft at the extracellular extremity. Surprisingly, the earlier identified active site serine and histidine are very far (~9 Å) apart, which questions their involvement in the same catalytic triad. Since, no unambiguous identification of the active site could be made, we investigated the roles of all acidic residues in activity (described in chapter 3). Three acidic residues (Asp83, Asp85 and Asp210) like His212 turned out to be essential for catalysis. Asp210 forms a couple with His212 on one side of the active site that faces the Asp83-Asp85 couple on the other side. We propose that these four residues constitute a catalytic site, which has not been observed in proteases before. Based on this, we propose a novel catalytic mechanism, which involves the activation of a water molecule by the His-Asp couple, similar to the activation of the serine in serine proteases. Furthermore, we propose that the Asp-Asp couple is involved in stabilization of the oxyanion, similar to which is proposed for aspartic proteases. We solved the crystal structure of OmpT in complex with an inhibitor, zinc (described in chapter 4). Although the zinc binding sites are not very specific, this structure does supports our hypothesis on the catalytic site. Also based on the structure of OmpT, and that of another OMP in complex with LPS, we identified an LPS-binding site in OmpT. Based on the location of this site, we conclude that LPS must have an indirect effect on catalysis, for example by inducing a slight conformational change. &#13;\nNspA of Neisseria meningitidis, the main cause of life-threatening meningitis, has been found to be well-conserved and to elicit bactericidal and protective antibodies in mice. These findings makes NspA a very attractive vaccine candidate. We solved the crystal structure of NpsA (described in chapter 5) in order to know the exact three-dimensional conformation of the loop (loop 3) against which the antibodies are directed. This conformation can form the basis for the design of cyclic peptides that adopt this conformation. These peptides may be used as vaccine against Neisseria meningitidis. The structure of NspA furthermore reveals a hydrophobic cleft with a detergent molecule bound. This might be related to the yet unknown function of NspA. &#13;\nIn conclusion, the structure NspA can be used as a basis for the design of a vaccine, and the structure of OmpT can form a basis for antimicrobial drug design.