Active site loop of metallo-beta-lactamases tunes metal site coordination and affects catalysis by second sphere interactions

MOJICA, M.F.; GIANNINI, E.; VILA, A.J.; TARACILA, M.A.; LLARRULL, L.I.; PALACIOS, A.R.; BETHEL, C.R.; BONOMO, R.A.

Florianópolis

Conferencia; ICBIC18 - International Conference on Biological Inorganic Chemistry; 2017

Beta-lactams are the most important class of clinically available antibiotics used to treat bacterial infections. The main beta-lactam resistance mechanism is the expression of beta-lactamases, enzymes that selectively hydrolyze beta-lactams. Among them, Metallo-beta-Lactamases (MbetaLs) are metal-dependent enzymes of particular concern given their ability to hydrolyze virtually all classes of beta-lactam antibiotics and cause to date clinically useful MbetaL inhibitors are not available[1]. NDM-1 is an enzyme that poses an increasing clinical interest since it is one of the most potent and widespread MbetaLs. It has been reported that the active site loop 3 (ASL-3) of MbetaLs plays an important role on substrate recognition[2]. Comparison of cristal structures of NDM-1 to other MbetaLs has revealed that its ASL-3 lacks a conserved Pro residue at its C-terminus leading to a more flexible loop and a wider active site[3].To test the hypothesis that these features account for the higher affinity and catalytic activity of NDM-1, we characterized three ASL-3 mutants of NDM-1, two in which the ASL-3 was replaced by those of two clinically relevant MbetaLs: IMP-1 (more charged) and VIM-2 (one residue shorter), and one in which we introduced the conserved Pro residue at the base of the loop. Biochemical characterization of these enzymes included minimum inhibitory concentration tests, steady state and pre-steady state kinetics against different beta-lactam antibiotics, thermal stability and analysis of active site coordination using Co(II) derivatives and x-ray crystallography. We found that modifications on the ASL-3 slightly affect the substrate spectra. Instead, it affects the global protein structure and stability and, surprisingly, the accumulation of mechanistic reaction intermediates. We interpret that these observations are due to alterations of the second shell residues of the metal site that function as a link between the engineered loop and the catalytic site. These are unprecedented findings in the structure-function relationship of MbetaLs showing the tight connection between the protein scaffold and the ASL-3 and disclosing a crucial role of this loop in their resistance profile.Acknowledgments: CONICET, ANPCyT and NIH.References[1] M. Mckenna, Nature, 499, 394-6 (2013)[2] T. Palzkill, Ann N Y Acad Sci, 1277, 91-104 (2013)[3] D. King and N. Strynadka, Protein Sci., 20, 1484-91 (2011)