NmerA of Tn501 Mercuric Ion Reductase: Structural Modulation of the pKa Values of the Metal Binding Cysteine Thiols,

摘要

To avoid nonspecific and/or undesirable binding and reactivity of metal ions with cellularncomponents, organisms have evolvedmetal-specific systems for trafficking proteins. Although systems differ,nthose handling soft metal ions such as Hg2þ,Cuþ,Zn2þ, etc., all utilize heavy metal-associated (HMA)nproteins and domains of ∼70 amino acids with a conserved GMXCXXC motif in a βRββRβ structural fold.nWhile the conserved cysteines define a common metal binding site in these proteins, other structural featuresnmust be utilized to create metal ion, protein partner, and contextual specificities. This paper presents initialnstructure-function studies of the N-terminal HMA domain (NmerA) of Tn501 mercuric ion reductasen(MerA) aimed at identifying structural features critical to its role in facilitating efficient transfer of Hg2þ tonthe MerA catalytic core for reductive detoxification. First, NMR solution structures of reduced and Hg2þ-nbound forms of NmerA are presented that allow definition and comparison of the structure of the metalnbinding loop in the two states. Structural differences between the two forms are compared with differencesnobserved in threeHMA domains with differentmetal ion and functional contexts. Second, analyses of theUVnabsorbance properties of wild-type, Cys11Ala, and Cys14Ala forms of NmerA are presented that providenassignments of the pKa values for the two cysteine thiols of the metal binding motif. Third, results from 13nC NMR studies with wild-type and Y62F NmerA labeled with [β-n13nC]cysteine are presented that define anrole for Tyr62 in modulating the pKa values of the cysteine thiols.