A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. β-lactam targeting of penicillin-binding protein (PBP)–mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA–PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA–PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria

Structures and ligand interactions of penicillin binding proteins in gram-negative bacteria

Antibiotic resistance has become one of the major threats to global health, because of the increasing emergence of multi-drug resistant bacteria and the lack of new effective antibiotics to counteract them. Therefore, new antibiotics need to be developed, and bacterial cell wall biosynthesis has long been recognised as an excellent target for antibacterial discovery. Transpeptidation is the last reaction in the synthesis of peptidoglycan and is catalysed by penicillin binding proteins, the enzymes targeted by β-lactam antibiotics. In this project, a novel crystal structure of PBP2 has been determined from Acinetobacter baumannii, a Gram-negative pathogen commonly causing nosocomial infections, to aid the structure-based design of new PBP-targeting molecules. Structures of PBP2 in complex with b-lactam antibiotics and diazabicyclooctane compounds have been also generated, highlighting particular protein-ligand interactions that could inform the optimisation of PBP inhibitors from existing and novel chemical classes. Moreover, the structure of A. baumannii PBP2 has disclosed distinctive features in the transpeptidase domain of this enzyme, including a new zinc-binding site proximal to the catalytic pocket, that is essential for the cell wall elongation in this pathogen. β-lactam molecules generated by the polymerisation of ampicillin in solution have been also investigated, with particular emphasis on the interactions with PBPs and β-lactamases. The covalent binding of ampicillin and its polymers to various PBPs from Gram-negative bacteria have been confirmed by mass spectrometry, and structural studies of Pseudomonas aeruginosa PBP1a and PBP3 have been undertaken to elucidate the binding mode of these particular molecules. Ampicillin and ampicillin polymers are hydrolysed by the β-lactamase AmpC at different rates, and they also show differences in antimicrobial activity. This study provides the structural basis for the inhibition of important HMW-PBPs by β-lactam and non-β-lactam compounds, and it could be useful for the development of potent PBP inhibitors

Histone deacetylases: Structural determinants of inhibitor selectivity

Histone deacetylases (HDACs) are epigenetic targets with an important role in cancer, neurodegeneration, inflammation, and metabolic disorders. Although clinically effective HDAC inhibitors have been developed, the design of inhibitors with the desired isoform(s) selectivity remains a challenge. Selective inhibitors could help clarify the function of each isoform, and provide therapeutic agents having potentially fewer adverse effects. Crystal structures of several HDACs have been reported, enabling structure-based drug design and providing important information to understand enzyme function. Here, we provide a comprehensive review of the structural information available on HDACs, discussing both conserved and isoform-specific structural and mechanistic features. We focus on distinctive aspects that help rationalize inhibitor selectivity, and provide structure-based recommendations for achieving the desired selectivity