NMR Solution Structure of the Oxidized Form of MerP, a Mercuric Ion Binding Protein Involved in Bacterial Mercuric Ion Resistance^,

Abstract

Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.

Molecular Analysis of the Copper-Transporting Efflux System CusCFBA of Escherichia coli

“…CusF could act as a periplasmic copper chaperone shuttling copper to the CusCBA efflux complex and thereby increasing the accessibility of periplasmic copper. This function is reminiscent of periplasmic substrate binding proteins that shuttle their substrates to the MerT transporter or ABC uptake systems (25,27,30,36,43,48,51,52). Another small periplasmic copper-binding protein, the CopC protein from P. syringae, binds Cu(I) and Cu(II) to different sites, and redox change of the bound copper ion causes its migration from one site of CopC to the other (6).…”

Reactivity of the Two Essential Cysteine Residues of the Periplasmic Mercuric Ion-binding Protein, MerP

“…A comparison of the structures of free and Hg 2ϩ -liganded MerP indicates that Cys-17 migrates to the surface of the protein, in the process partially unravelling the amino terminus of the ␣-helix of which it is a part (6); a similar structural change is observed after formation of MerP ox (36). A possible mechanism for this might be that Hg 2ϩ initially binds to Cys-14 at the surface of the protein and then attracts the negatively charged thiol from its inaccessible position.…”

“…The lowering to 5.5 of the pK a of the Cys-17 thiol from a more typical 8.7 would be expected to stabilize MerP red by 4.3 kcal/ mol, which is greater than the observed value of 2.7 kcal/mol. However, the conformational change in going from reduced to oxidized MerP involves movement of Cys-17 from a buried to exposed position and other associated structural changes that amount to more than simple removal of a thiolate (6,36). Thus, the low pK a of Cys-17 is not the sole determinant of the relative stabilities of MerP ox and MerP red .…”

“…Stabilization of the negatively charged thiolate anion by the helix dipole lowers the pK a (41), but in model ␣-helical peptides this can only account for a lowering of the thiol pK a by up to 1.6 units (42). It is interesting to note that it is only in the reduced form of MerP that Cys-17 is part of the ␣-helix, whereas in the oxidized and Hg 2ϩ -bound forms the ␣-helix starts at residues 18 and 19, respectively (6,36). A second thiolate-stabilizing feature of both thioredoxin and DsbA is hydrogen bonding of backbone amides to the sulfur of the low-pK a thiol (43,44).…”

Measurement of volatilized mercury by a mini-system: a simple, reliable and reproducible technique

“…This mechanism is of interest since it codes proteins for transport and reduction of mercuric ions in Gram-negative bacteria. (Silver and Phung, 1996;Quian et al, 1998;Nies, 1999). Resistance to mercury is based on particularities of the metal such as its redox potential, vapor pressure, and low melting and boiling point.…”