Single Mutation Distal from the Active Site Optimized Zn(II) Affinity in the NDM Carbapenemase

DELMONTI, JULIANA.; VILA, ALEJANDRO J.; MORGADA, MARCOS M.

Interlaken

Conferencia; 19th International Conference on Biological Inorganic Chemistry; 2019

The New Delhi metallo-ß-lactamase (NDM) is a periplasmatic Zn(II)-dependent enzyme capable to hydrolyze carbapenems, the last resort antibiotic against multiresistant bacteria. During infection the host immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. This impacts directly on periplasmatic Zn(II) levels. In metal limitation conditions, metallo-ß-lactamases (MBL), and in particular NDM, lose activity against ß-lactams by dissociation of the Zn(II) cofactor, and the periplasmic accumulation in vivo of the enzymes decreases. Since its discovery in 2008, NDM has shown a fast worldwide spread with 27 natural variants reported in the clinic. NDM variants differ by a few mutations outside the active site, with the substitution M154L being the most frequent. Studies in our group have shown that the tolerance to Zn(II) starvation is selected during the evolution of NDM. It has been reported that the mutation M154L increases the resistance under zinc deprivation conditions in vivo without imparting protein stabilization. Moreover, all double mutants containing M154L substitution have shown the highest resistance under Zn(II) deprivation conditions. Between them, the variant NDM-15 with mutations M154L and A233V was the least susceptible to metal depletion.In this way, we focus on the substitution M154L present on the natural variant NDM-4 as a single mutation and more than the half of the alleles. We aim to assess the biochemical and biophysical features that are tailored by the mutation to endure the action of the immune system response. First, we measured the stability of the purified variants and their apo (non-metalleted) derivatives by thermal shift analysis. Alleles NDM-4 and NDM-15, presented slightly higher Tm values than NDM-1. Nevertheless, they all displayed the same Tm gap between holo (metallated) and apo forms. These results indicate that the mutation M154L does not increase significantly protein stability. We performed competition experiments with Zn(II) chromophoric chelators. We proved that substitution M154L impacts significantly on the first binding event, without changing the affinity of the second Zn(II) ion. By UV-Vis spectroscopy and paramagnetic NMR studies with Co(II) substituted variants we demonstrated that the increment in Zn(II) affinity is not due to changes on metal coordination in the active site. 15N-1H HSQC spectra of holo NDM-4 and NDM-1 were almost superimposable, indicating that the mutation does not affect significantly the backbone structure in solution as expected from the reported Xray crystal structures. However, the HSQC spectrum of apo NDM-4 showed a lower amount of signals due to line broadening compared to apo NDM-1, reflecting more structural flexibility of apo NDM-4.Overall, these results suggest that mutation M154L might be altering the dynamic or accessibility of residues near the active site in the apo-form consequently affecting metal affinity of the protein, mainly changing the first Zn(II) binding event.