Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils

Abstract

Arsenic, a contaminant of water supplies worldwide, is one of the most toxic inorganic ions. Despite arsenic's health impact, there is relatively little structural detail known about its interactions with proteins. Bacteria such as Escherichia coli have evolved arsenic resistance using the Ars operon that is regulated by ArsR, a repressor protein that dissociates from DNA when As(III) binds. This protein undergoes a critical conformational change upon binding As(III) with three cysteine residues. Unfortunately, structures of ArsR with or without As(III) have not been reported. Alternatively, de novo designed peptides can bind As(III) in an endo configuration within a thiolate-rich environment consistent with that proposed for both ArsR and ArsD. We report the structure of the As(III) complex of Coil Ser L9C to a 1.8-Å resolution, providing x-ray characterization of As(III) in a Tris thiolate protein environment and allowing a structural basis by which to understand arsenated ArsR.arsenic-binding proteins ͉ coiled coil peptides ͉ crystallography ͉ heavy metal toxicity ͉ protein design A rsenic toxicity is a worldwide problem as a natural contaminant of water supplies. Despite the fact that it is a human toxin and carcinogen, few structural reports on its interaction with biological ligands have appeared. Escherichia coli and other bacteria have evolved a detoxification mechanism that employs the arsRDABC operon (1). Arsenic removal by these encoded proteins is initiated when As(III) binds to ArsR, resulting in the dissociation of the repressor protein from the promoter DNA. The structure of the ArsA component of the ATP-dependent extrusion pump ArsAB with antimonite bound in close proximity to the nucleotide-binding site was able to provide some insight into the active transport of As(III) out of the cell (2). Additionally, structural characterization of substrate and product complexes of the arsenate reductase ArsC, which reduces arsenate (AsO 4 3Ϫ ) to arsenite (AsO 2 Ϫ ), has helped elucidate this step of the arsenic detoxification pathway (3, 4). Recent studies of E. coli ArsD indicate that it is a metallochaperone, transporting As(III) to ArsA for extrusion (5). Extended x-ray absorption fine structure and mutagenesis studies have shown that ArsR coordinates As(III) with three cysteine thiolates at a distance of 2.25 Å, a coordination mode that ArsD is also proposed to employ (1, 5). However, no x-ray or NMR structures of ArsR, the repressor protein, or ArsD have been reported to date. Furthermore, AsS 3 structures have not been reported for any biologically relevant small-molecule thiolates, such as glutathione, and only a handful of related AsS 3 complexes with aromatic ligands or chelated alkyl dithiolate coordination are reported (6).We set out to model the putative As(III) coordination environments of ArsR and ArsD in a designed peptide system. Previously, we have shown that the three-stranded coiled coildesigned with the heptad repeat strategy, such as TRI L16C (sequences of all peptides are in Table 1...

Using diastereopeptides to control metal ion coordination in proteins

“…Substitution of either an a or d Leu with Cys provides a preorganized homoleptic thiol site in the interior of these coiled coils. The binding of numerous heavy metals such as Hg(II), Bi(III), Pb(II), and As(III) has been explored (12)(13)(14)(15)(16)(17)(18)(19), although the most intriguing system has been Cd(II) that binds to TRIL16C as a mixture of 3-and 4-coordinate CdS 3 and CdS 3 O (O from an exogenous water molecule) forms, respectively.…”

Natural and Artificial Proteins Containing Cadmium

“…Cys containing analogues of TRI have previously been shown to be capable of binding to various heavy metal ions including Hg(II), Bi(III), As(III), and Pb(II) [38][39][40][41][42][43][44][45][46][47][48]. However, two options exist for introducing a Cys residue into the coiled coil interior as Leu side chains in both the a and d sites are directed towards the hydrophobic core.…”

Maquette Strategy for Creation of Light- and Redox-Active Proteins

“…Early demonstrations showed iron sulfur clusters spontaneously assemble in maquettes 2 singly or in pairs, with or without heme(s); later singlechain variants were designed [Gibney et al, 1996;Mulholland et al, 1998;Musgrave et al, 2002;Nanda et al, 2005;Grzyb et al, 2010;Nanda et al, 2016]. Maquette frames of the kind in Figure 7 have also proved to offer a creative environment for metals alone and in clusters ( Figure 5C) [Dieckmann et al, 1997;Farrer et al, 2000;Calhoun et al, 2005;Geremia et al, 2005;Touw et al, 2007;Calhoun et al, 2008;Tegoni et al, 2012;Zastrow et al, 2012;Roy et al, 2014].…”