The Characterization and Structure of the Manganese-responsive Transcriptional Regulator ScaR†§
| Autor: | William E. Draper, Wendy A. Breyer, Rebecca K. Phillips, Kate E. Stoll, Seth M. Cohen, Richard G. Brennan, Howard F. Jenkinson, Joseph I. Kliegman, Hattie K. Brown, Rhoda A. T. Brew-Appiah, Nicholas S. Jakubovics, Misha V. Golynskiy, Arthur Glasfeld |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Base pair
Operon Molecular Sequence Data Repressor ATP-binding cassette transporter Plasma protein binding Biology Crystallography X-Ray Biochemistry Article Bacterial Proteins Nickel Amino Acid Sequence Binding site Peptide sequence Manganese Sequence Homology Amino Acid Streptococcus DNA-binding domain Cobalt DNA Gene Expression Regulation Bacterial Zinc Chromatography Gel Protein Multimerization Ultracentrifugation Cadmium Protein Binding |
| Popis: | Manganese is an essential nutrient for many pathogenic bacteria and has been linked to virulence in a number of species (1–3). A cofactor for a variety of enzymes involved in fighting oxidative stress, manganese is taken up in bacteria by NRAMP and ABC-type transporters. In streptococci the lipoprotein receptor antigen I (LraI) family of proteins have been linked to infection and have been identified as periplasmic manganese-binding proteins, functioning as a component in ABC transporters. In addition, LraI proteins in streptococcal species are important for virulence and are possible vaccinogens against endocarditis (3, 4). In Streptococcus gordonii, an oral bacterium that colonizes the mouth and can cause endocarditis, an LraI homolog plays a role in interbacterial cell adhesion among colonies of oral bacteria (5). Referred to as ScaA (for streptococcal coaggregation adherance), it is part of an inducible high affinity ABC transporter of Mn2+ encoded by the scaCBA operon (6). The expression of Mn2+-selective ABC transporters in streptococci is under the regulatory control of a group of homologous metalloregulatory proteins, including ScaR from S. gordonii, SloR from S. mutans, PsaR from S. pneumoniae and MtsR from S. pyogenes (4, 7–9). They share greater than 50% sequence identity and are either Mn2+-specific or give a mixed response to iron and manganese (6, 8). These regulators are essential to maintaining manganese homeostasis, and loss of the regulator has been shown to impair the virulence of S. pneumoniae (9). The first member of this group to be characterized, ScaR, selectively represses expression of the scaCBA operon in the presence of its corepressor manganese (7). Elevation of manganese concentration from 0.1 to 50 µM decreases ScaA production five-fold. Gel shift assays performed with ScaR and DNA fragments containing the scaC promoter region showed that Mn2+, and to a lesser extent Zn2+ and Ni2+, activate ScaR for DNA-binding, while Fe2+, Cu2+ and Co2+ are less effective at 100 µM concentrations (7). ScaR protects a 46-base pair stretch of DNA in the promoter region. Two adjacent binding sites for the repressor, each comprised of inverted repeats, were identified based on sequence alignment to a number of other promoter regions from related operons in streptococcal species (7). ScaR and its streptococcal homologs belong to the DtxR/MntR family of metalloregulatory proteins. The namesakes of this family regulate iron (DtxR) and manganese (MntR) uptake in Corynebacterium diphtheriae and Bacillus subtilis respectively (10, 11). Both DtxR and MntR are dimers in their active states, but despite similar structures and a number of conserved metal-binding residues in the two proteins, each is activated by metal ions in a distinct manner. DtxR and its closely related homolog, IdeR from M. tuberculosis (Figure 1), are functional dimers of three-domain subunits that are activated by metal binding to two sites in each subunit 9 A apart (12–14). The so-called primary site is located at the interface of the N-terminal DNA binding domain and the central dimerization domain, and the ancillary site is at the interface of the dimerization domain and a C-terminal domain of unknown function. In contrast, MntR is a two-domain protein, lacking the C-terminal domain found in DtxR, and binds two metal ions 4.4 A apart, at sites labeled A and C, in a binuclear complex at the interface of the DNA-binding and dimerization domains (Figure 1; 15, 16). The selectivity of DtxR for iron over manganese derives from ligand selection (17). The primary site in DtxR contains two sulfur-containing residues, Met10 and Cys102, while no known proteins selective for Mn2+ are known to use sulfur atoms as ligands. In MntR, metal-ion selectivity is proposed to derive from coordination geometry of the A site, which appears to select for divalent cations with relatively large ionic radii (16). Such binding assists in the formation of the necessary geometry in the C site, allowing the second metal ion to bind and activating MntR for DNA binding. Activated DtxR binds to its cognate DNA sequences as a pair of dimers on opposing faces of the duplex, contacting DNA over a region of approximately 27 base pairs (18). Although the interaction of MntR with its cognate DNA sequences is not defined, its cognate operator sequence is roughly 20 base pairs long (11). Figure 1 Structures of (A) IdeR bound to Co2+ (PDB ID 1F57; 32) and (B) MntR bound to Mn2+ (PDB ID 2F5D; 16). Side chains for metal binding residues are shown for one subunit of each dimer as are bound metal ions (in pink). The N-terminal, DNA-binding domains ... The origins of specificity of ScaR for its cognate metal ion co-repressor and for its operator sequence(s) are unknown. Structurally, ScaR is more similar to the iron regulator, DtxR, than the manganese regulator, MntR. Though sharing only 24% sequence identity, ScaR preserves the three domain structure of DtxR and many of the metal-binding residues found in DtxR. Yet ScaR is manganese-specific while DtxR in iron-specific (7, 17). Ligand selection may play a role, as the DtxR metal-binding residues Met10 and Cys102 are Asp7 and Glu99 at analogous positions in ScaR. Also, sequence comparisons indicate that ScaR may bind metals in a fashion distinct from DtxR, as two of the ancillary site residues in DtxR, Glu173 and Gln175, do not appear to be present in ScaR. Furthermore the interaction of ScaR with the scaC promoter region appears to be novel within the DtxR/MntR family, with two non-overlapping recognition sites (7). In this work, we present results from crystallographic and solution studies that ScaR binds metal ions and DNA in a different fashion than DtxR or MntR, and represents a new subclass of metal-dependent regulators in the DtxR/MntR family. |
| Databáze: | OpenAIRE |
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