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Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Published on: June 28, 2013
Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic
1Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CNRS/Commissariat à l'Energie Atomique), 41, rue Jules Horowitz, 38027 Grenoble, France. dessen@ibs.fr
Abstract:
Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.
Insights
Penicillin-binding proteins (PBPs) are key targets for beta-lactam antibiotics. Mutations in Streptococcus pneumoniae PBP2x create an "open" active site, facilitating resistance by altering substrate recognition.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Penicillin-binding proteins (PBPs) are crucial targets for beta-lactam antibiotics in bacteria.
- Antibiotic resistance, particularly in Gram-positive bacteria like Streptococcus pneumoniae, often arises from modifications in PBP genes.
- PBP2x is a key protein conferring high-level resistance to beta-lactams in S. pneumoniae.
Purpose of the Study:
- To elucidate the atomic-level mechanisms of beta-lactam resistance in Streptococcus pneumoniae.
- To understand how mutations in PBP2x contribute to penicillin resistance.
Main Methods:
- X-ray crystallography was used to determine the structure of PBP2x from a resistant S. pneumoniae isolate (Sp328).
- Analysis of mutations within the PBP2x active site and their impact on protein structure and function.
Main Results:
- The crystal structure of Sp328 PBP2x, containing 83 mutations, was solved.
- Specific mutations (Thr338Ala, Ser389Leu, Asn514His) were identified near the active site.
- These mutations weaken hydrogen bonds, displace a key water molecule, and create a more "open" active site conformation.
Conclusions:
- The structural alterations in PBP2x lead to an "open" active site.
- This open conformation may allow the enzyme to bind and process abnormal, branched peptidoglycan substrates characteristic of resistant strains.
- Understanding these structural changes is vital for developing new strategies against beta-lactam-resistant bacteria.
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