Structural basis of antibiotic recognition in the metallo-β-lactamase NDM-1

MOJICA, MARÍA F.; LLARRULL, LETICIA I.; PALACIOS, ANTONELA R.; KLINKE, SEBASTIÁN; VILA, ALEJANDRO J.; GIANNINI, ESTEFANÍA ; OTERO, LISANDRO HORACIO; BONOMO, ROBERT

San Luis

Congreso; XII Reunión Anual de la Asociación Argentina de Cristalografía; 2016

Asociación Argentina de Cristalografía

β-Lactam antibiotics remain the most useful chemotherapeutic agents in the fight against bacterial infections. Despite much progress in antibiotic design throughout six decades, resistance to β-lactams is now a serious clinical problem. The most prevalent resistance mechanism is the expression of bacterial enzymes tailored to hydrolyze these antibiotics: β-lactamases. Among them, Metallo-β-lactamases (MβLs) are an important public health issue because they are able to efficiently hydrolyze almost all β-lactams, including the carbapenems considered as ?last resort? antibiotics. Moreover, there is no clinically useful inhibitor for MβLs[1]. Clinically relevant MβLs have two catalytically essential Zn(II) ions at their active sites, a conserved metal ligand set and a common catalytic mechanism (Figure 1). Despite showing low sequence identity, MβL structures reveal a common protein fold (αβ/βα) and their active sites are superimposable[2]. It has been shown that differences in substrate profiles among MβLs are highly dependent on the active site environment, mainly on second shell residues and loops that shape the active site [3]. In this context we are trying to unveil the structural features that determine the substrate profile of the MβL NDM-1, evaluating the role of some residues from its active site environment, through second shell mutation and loops engineering. The crystal structure of NDM-1 revealed that loop L3 is more open, flexible and hydrophobic, giving rise to a more open active site than in other MβLs[4]. To analyze the importance of these loop features, we have generated a mutant carrying the loop of another MβL (IMP-1), which contains many charged residues and also a proline residue at the base of L3, which is conserved in other MβLs and may lead to a decrease in loop flexibility[5]. We were able to purify the mutant (NDM-1_loopIMP), screened different crystallization conditions with a Gryphon robot and obtained good quality crystals after optimization. Preliminary X-ray diffraction data were obtained at the Leloir Institute (PLABEM) with a Bruker D8 QUEST microfocus source, and the final datasets were collected at the SOLEIL Synchrotron (France).We were able to trace all residues in the loop and refine the structure to 1.65 Å. These results allowed us to gain a more complete understanding of the structural bases of antibiotic recognition in NDM-1.