Unprecedented allosteric inhibition of E. coli malate dehydrogenase by silver(I) from atomic resolution view

Abstract

Metal ions Metal ions may functionally inhibit metalloproteins via either replacement of intact metal cofactors or binding to allosteric sites via metalloallostery. Despite extensive studies, up to now, it is not fully understood how silver inhibits its authentic protein targets, particularly at the atomic level, largely owing to the lack of knowledge on silver authentic protein targets as well as limited structures available. Herein we show that malate dehydrogenase (MDH) serves as a vital target of antimicrobial Ag+ against E. coli. Ag+ binds MDH at multiple sites and inhibits its activity via a non-competitive mechanism. Importantly, we successfully captured Ag+-mediated “open-to-closed” conformational change of the active-site of MDH by X-ray crystallography. Combined with the enzyme kinetics and mutagenesis data, we unambiguously unveil that the allosteric inhibition of MDH by Ag+ is attributable to its binding to the cysteine residue (Cys251), consequently leading to the closure of the active-site loop of MDH, which disrupts the substrate and coenzyme binding, and ultimately inhibiting the activity of MDH. Our studies provide the first structural glimpse of an unprecedented allosteric inhibition of authentic target enzymes by silver. These findings not only enhance our understanding on the mechanism underlying silver inhibition of its protein targets at the atomic level, but also offers a novel allosteric targeting site in MDH for the design of new antibiotics.