Abstract
Metal ions may functionally inhibit metalloproteins via either replacement of intact metal cofactors or binding to allosteric sites via metalloallostery. Despite extensive studies, until now, it has not been fully understood how silver inhibits its authentic protein targets, particularly at the atomic level, largely owing to the lack of knowledge on the authentic protein targets of silver as well as the 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 the 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 of the mechanism underlying silver inhibition of its protein targets at the atomic level, but also offer a novel allosteric targeting site in MDH for the design of new antibiotics.