The Crystal Structure of Zn(II)-Free Treponema pallidum TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation

Abstract

We previously demonstrated that Treponema pallidum TroA is a periplasmic metal-binding protein (MBP) with a distinctive alpha-helical backbone. To better understand the mechanisms of metal binding and release by TroA, we determined the crystal structure of the apoprotein at a resolution of 2.5 Å and compared it to that of the Zn(II)-bound form (Protein Data Bank accession code 1toa). apo-TroA shows a conformation even more closed than that of its Zn(II)-bound counterpart due to a 4°tilt of the C-terminal domain (residues 190 through 308) about an axis parallel to the poorly flexible backbone helix. This domain tilting pushes two loops (residues 248 through 253 and 277 through 286) towards the metal-binding site by more than 1 Å, resulting in an unfavorable interaction of I251 with D66. To avoid this contact, D66 shifts towards H68, one of the four Zn ( Originally reported to be an outer membrane protein of the syphilis spirochete Treponema pallidum (3, 4), TroA has been shown by genetic, ultrastructural, and X-ray crystallographic studies to actually be the binding component of an ATP-binding cassette (ABC) transport system which shuttles Zn(II) and possibly other transition metals (e.g., manganese) across the T. pallidum cytoplasmic membrane (1, 11-13, 18, 21; K. R. O. Hazlett, F. Rusnak, and J. D. Radolf, unpublished observations). TroA is 1 of more than 40 members of a newly defined cluster of metal-binding proteins within the extended family of bacterial solute-binding receptors (SBRs) (8,23).Members of the SBR family have N-and C-terminal domains separated by a crevice which serves as the ligand-binding site (22). The domains of the receptors for nonmetal ligands such as sulfate, phosphate, amino acids, oligopeptides, monosaccharides, and oligosaccharides are hinged by two or three ␤ strands that function according to a so-called "Venus flytrap" mechanism. According to this model, a hinge-bending motion traps the ligand within the cleft between the two domains. Conversely, upon release of the ligand, the receptor adopts an open conformation, in which the two domains of the apoprotein are further apart and the binding site is more solvent accessible (22). Unlike with the nonmetal SBRs, the two domains of the MBPs are linked by an extended and presumably poorly flexible ␣-helix which interacts along its entire length with both domains (17, 18). Because of this structural difference, we hypothesized that ligand binding and release by MBPs would proceed via mechanisms different from those by which non-metal-binding SBRs proceed. We have obtained evidence for this hypothesis by determining the crystal structure of apo-TroA and comparing it to that of the Zn(II)-bound form of the protein.Preparation and crystallization of apo-TroA. A recombinant DNA construct encoding TroA with an N-terminal His 6 tag but without its leader peptide (residues 23 through 308) 6 was previously described (11). Protein was overexpressed in Escherichia coli DH5␣ and purified from cell lysates by use of a Ni-nitrilotriacetic acid agaros...

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus

“…This structure is thought to result in an inability of these proteins to undergo large scale conformational changes upon ligand binding. Indeed, high resolution structures of E. coli BtuF and Treponema pallidum TroA, in both the liganded and unliganded forms, indicate this is to be the case (32)(33)(34). Our results indicate that, in solution, both FhuD1 and FhuD2 from S. aureus do not demonstrate a ligand-induced, large scale conformational change, in agreement with high resolution crystal structures of other members of this binding protein family.…”

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

“…2. This rotation is quite modest compared with other PBPs but much larger than the 4°o bserved in the case of the zinc-binding protein TroA (25,26). The hinge motion in NikA is similar to those in OppA and DppA but smaller (30,48).…”

“…Unlike the ␤ strands, which connect the lobes of most solute-binding proteins, the helix is rigid and permits only a very small conformational change on ligand binding. It has been suggested that this is necessary for tight metal binding given the small ligand size (26). The crystal structure of a molybdate-binding protein from Azotobacter vinelandii, however, shows a more usual topology with a flexible hinge (28).…”

“…The crystal structures of two metal binding proteins in the solute-binding protein family, however, show a very different connection between the two domains, forming a new family of metal binding receptors (24). TroA is a zinc-binding protein from Treponema pallidum (25,26), and PsaA is a manganese/zinc-binding protein from Streptococcus pneumoniae (27). In these proteins, the polypeptide chain passes from one domain to the other only once, this connection being a long helix running most of the length of the protein.…”

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

“…6803 MntC (Syn-MntC) [12], and Eco-ZnuA [13,14] as well as the metal-free (apo) structures of Tpa-TroA [15] and mutant Syn-ZnuA (without the His-rich loop) [8] have been determined by X-ray crystallography. The overall architecture of these MBRs is similar to that of other PLBPs from ABC transporter systems.…”