The Core Metal-Recognition Domain of MerR

Abstract

MerR, the metalloregulatory protein of the mercury-resistance operon (mer) has unusually high affinity and specificity for ionic mercury, Hg(II). Prior genetic and biochemical evidence suggested that the protein has a structure consisting of an N-terminal DNA binding domain, a C-terminal Hg(II)-binding domain, and an intervening region involved with communication between these two domains. We have characterized a series of MerR deletion mutants and found that as little as 30% of the protein (residues 80-128) forms a stable dimer and retains high affinity for Hg(II). Biophysical measures indicate that this minimal Hg(II)-binding domain assumes the structural characteristics of the wild-type full-length protein both in the Hg(II) center itself and in an immediately adjacent helical protein domain. Our observations are consistent with the core Hg(II)-binding domain of the MerR dimer being constituted by a pair of antiparallel helices (possibly in a coiled-coil conformation) comprised of residues cysteine 82 through cysteine 117 from each monomer followed by a flexible loop through residue cysteine 126. These antiparallel helices would have a potential Hg(II)-binding site at each end. However, just as in the full-length protein, only one of these potential binding sites in the deleted proteins actually binds Hg(II).

Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

“…Protein concentrations were routinely measured by the Bradford assay, with bovine serum albumin (BSA) as the standard protein, and were corrected to their OD 280 concentrations; for full-length MerR, the BSAstandardized Bradford assay correction factor was 1.024, and for MBD, it was 1.487. It was noted previously that the BSA-standardized Bradford assay underestimates protein concentrations for truncated MerR derivatives (31).…”

“…The relative affinities of MBD and MerR for Hg(II) were determined by conventional free dialysis and by equilibrium ultrafiltration (31). For equilibrium ultrafiltration, 10.0 M protein in 50 mM Tris-HCl (pH 7.9) buffer containing 500 mM NaCl, 5 mM BME, and 10% glycerol was incubated with 5 to 20 M 203 HgCl 2 for 15 min at room temperature.…”

“…The cellular content of MerR or MBD was quantified by Western blotting (see above). (31) indicated that its Hg(II)-binding ability lies in residues 82 to 126. MBD was designed as a single polypeptide that could fold into an antiparallel coiled coil, thereby forming a structure like that formed by the two chains of the MerR dimer (Fig.…”

“…Three conserved cysteine residues in the Hg(II) binding domain of the MerR dimer form a trigonal Hg(II)-coordination site, employing C117 and C126 from one monomer and C82 from the other. Although a MerR dimer has two potential Hg(II)-coordination sites, it only binds one Hg(II) in vitro (23,(29)(30)(31).…”

“…The metal-recognition domain of these proteins is an antiparallel, coiled coil lying in the C-terminal half of the protein, as predicted by earlier genetic, biochemical, and biophysical work. Specifically, this antecedent work established that (i) the Hg(II) binding response of MerR requires three cysteines (C82, C117, and C126) and the protein functions as a dimer, employing C117 and C126 from one monomer and C82 from the other to bind Hg(II) (14,19,23); (ii) MerR has a long ␣-helix (residues 82 to 117) with a strongly predicted propensity to form a coiled coil (5,31); (iii) a deletion mutant containing only residues 80 to 128 of MerR folds into an ␣-helix and forms stable dimers capable of binding Hg(II) even in the presence of excess thiols (31); and (iv) crystal structures of two MerR family members, the aromatic drug-binding regulators BmrR and MtaN (11,13), show antiparallel coiled coils in regions corresponding to the C terminus of the metal-binding members of the MerR-family.…”