UBC Theses and Dissertations
UBC Theses and Dissertations
Crystallographic studies of bacterial proteins involved in β-lactam resistance Lim, Daniel C.
The β-lactams are an important class of antibiotics that target penicillin binding proteins (PBPs), bacterial enzymes that catalyze the final step of cell wall synthesis. The bacterial cell wall maintains cell shape and provides protection against osmotic lysis. PBPs catalyze the formation of peptide crosslinks in peptidoglycan, which are essential to the structural integrity of the cell wall. Due to the uniqueness of the peptidoglycan of bacterial cells (with no biochemical equivalent in humans), β-lactams have been highly successful in treating bacterial infections. Unfortunately bacteria have developed multiple resistance mechanisms that have substantially reduced the effectiveness of this class of antibiotics. Three main mechanisms of β-lactam resistance have been found in resistant bacteria: production of β-lactamase enzymes that hydrolyze and inactivate β-lactams, over-expression of multi-drug efflux pumps in Gram-negative bacteria that actively pump out β -lactams and other antibiotics, and the expression of resistant PBPs with unusually low affinities for β-lactams. The overall goal of this thesis is to use the technique of X-ray crystallography for the structural characterization of specific proteins involved in each of these three main mechanisms. PSE-4 is a class A β-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild type PSE-4 carbenicillinase has been determined to 1.95 Å resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. Most carbenicillinases are unique among class A β-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50 fold reduction in kcat/Km and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 Å resolution with the wild type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A β-lactamase structures. The molecular modelling studies presented here suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin a-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild type PSE-4 may be involved in accommodating this shift in the bound substrate position. MexR is a member of the MarR family of bacterial transcriptional regulators and is the repressor for the MexAB-OprM operon, which encodes a tripartite multidrug efflux system in Pseudomonas aeruginosa. Mutations in MexR result in increased resistance to multiple antibiotics due to over-expression of this efflux system. The crystal structure of MexR has been determined to 2.1 Å resolution in the absence of effector. The four copies of the MexR dimer in the asymmetric unit are observed in multiple conformations. Analysis of these conformational states in the context of a model of the MexR-DNA complex proposed in this study suggests that an effector-induced conformational change may inhibit DNA binding by reducing the spacing of the DNA binding domains. The inhibited conformation is exhibited by one of the four MexR dimers, which contains an ordered C-terminal tail from a neighbouring monomer inserted between its DNA binding domains and which is proposed to resemble the MexR-effector complex. The results of this study indicate that MexR may differ from the other described member of this family, MarR, in the nature of its effector, mode of DNA binding and mechanism of regulation. The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad spectrum β-lactam resistance in MRSA strains is the penicillin binding protein 2a (PBP2a). Due its low affinity for β-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at β-lactam concentrations which inhibit the β-lactam sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 Å resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefm, penicillin G and methicillin show for the first time β-lactam binding by a resistant PBP. An analysis of the PBP2a active site reveals the structural basis of its resistance and identifies features in β-lactams important for high affinity binding.
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