Single mutation distal from the active site optimized Zn(II) affinity in the NDM carbapenemase.

MORGADA, MARCOS N.; A. J. VILA; DEL MONTI, JULIANA

Congreso; ICBIC 2019; 2019

The New Delhi metallo-ß-lactamase (NDM) is aperiplasmatic Zn(II)-dependent enzyme capable to hydrolyze carbapenems, the lastresort antibiotic against multiresistant bacteria. During infection the host immuneresponse withholds nutrient metal ions from microbial pathogens by releasingmetal-chelating proteins such as calprotectin. This impacts directly on periplasmaticZn(II) levels. In metal limitation conditions, metallo-ß-lactamases (MBL), andin particular NDM, lose activity against ß-lactams by dissociation of theZn(II) cofactor, and the periplasmic accumulation in vivo of the enzymesdecreases.Since its discovery in 2008, NDM has shown a fast worldwidespread with 27 natural variants reported in the clinic. NDM variantsdiffer by a few mutations outside the active site, with the substitution M154L beingthe most frequent. Studies in our group haveshown that the tolerance to Zn(II) starvation is selected during theevolution of NDM. It has been reported that the mutation M154L increases the resistanceunder zinc deprivation conditions in vivowithout imparting protein stabilization. Moreover, all double mutantscontaining M154L substitution have shown the highest resistance under Zn(II)deprivation conditions. Between them, the variant NDM-15 with mutations M154Land A233V was the least susceptible to metal depletion.Inthis way, we focus on the substitution M154L present on the natural variantNDM-4 as a single mutation and more than the half of the alleles. We aim toassess the biochemical and biophysical features that are tailored by themutation to endure the action of the immune system response. First, we measuredthe stability of the purified variants and their apo (non-metalleted)derivatives by thermal shift analysis.  Alleles NDM-4 and NDM-15, presented slightlyhigher Tm values than NDM-1. Nevertheless,they all displayed the same Tm gap between holo (metallated) and apoforms. These results indicate that the mutation M154L does not increasesignificantly protein stability. Weperformedcompetition experiments with Zn(II) chromophoric chelators. We proved thatsubstitution M154L impacts significantly on the first binding event, withoutchanging the affinity of the second Zn(II) ion. By UV-Vis spectroscopy andparamagnetic NMR studies with Co(II) substituted variants we demonstrated that theincrement in Zn(II) affinity is not due to changes on metal coordination in theactive site.15N-1HHSQC spectra of holo NDM-4 and NDM-1 were almostsuperimposable, indicating that the mutation does not affect significantly the backbonestructure in solution as expected from the reported X-ray crystal structures. However, theHSQC spectrum of apo NDM-4 showed a lower amount of signals due to linebroadening compared to apo NDM-1, reflecting more structuralflexibility of apo NDM-4.Overall, these results suggest thatmutation M154L might be altering the dynamic or accessibility ofresidues near the active site in the apo-form consequently affecting metalaffinity of the protein, mainly changing the first Zn(II) binding event.