Characterization and Structure of the Manganese-Responsive Transcriptional Regulator ScaR^,

Abstract

The streptococcal coaggregation regulator (ScaR) of Streptococcus gordonii is a manganese-dependent transcriptional regulator. When intracellular manganese concentrations become elevated, ScaR represses transcription of the scaCBA operon, which encodes a manganese uptake transporter. A member of the DtxR/MntR family of metalloregulators, ScaR shares sequence similarity with other family members, and many metal-binding residues are conserved. Here, we show that ScaR is an active dimer, with two dimers binding the 46-bp scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd2+, Co2+ and Ni2+, but not Zn2+. The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homolog, the iron-responsive regulator DtxR. While each DtxR subunit binds a metal ion in two sites, labeled primary and ancillary, crystal structures of ScaR determined in the presence of Cd2+ and Zn2+ show only a single occupied metal binding site that is novel to ScaR. The site analogous to the primary site in DtxR is unoccupied, and the ancillary site is absent from ScaR. Instead, metal ions bind to ScaR at a site labeled “secondary”, which is composed of Glu80, Cys123, His125 and Asp160 and lies roughly 5 Å away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologs are activated by a mechanism distinct from that of either DtxR or MntR.

Solution Structure of Escherichia coli FeoA and Its Potential Role in Bacterial Ferrous Iron Transport

“…These results revealed similarity to streptococcal coaggregation regulator (ScaR) (PDB ID 3hru) (Z-score, 7.1), iron-dependent regulator (IdeR) (PDB ID 1u8r) (Z-score, 6.7), and diphtheria toxin repressor protein (DtxR) (PDB ID 1c0w) (Z-score, 5.6). These proteins are all part of the metal and DNA-binding protein families that function as transcriptional regulators (61)(62)(63)(64)(65)(66)(67)(68). In addition, these proteins all have three domains: a DNA-binding, a dimerization, and an SH3-like domain (64,69,70), where the structure of FeoA resembles the last domain (see Fig.…”

“…5B). The presence of metal ions signals these proteins to transition between the DNA-binding and nonbinding forms (61)(62)(63)(64)(65)(66)(67)(68)(69)(70)73). The SH3-like domains of IdeR and DtxR also play an important role in this conversion apart from sole metal binding.…”

“…Examining our solution NMR studies, done at high protein concentrations, has shown that FeoA is a monomeric protein, making it unlikely that it dimerizes noncovalently in a similar fashion to IdeR for function. Of the three homologues, the SH3-like domain of ScaR is only known to bind metal, with no specific function like the other two (64,68). FeoA shares greater structural similarity between all three C-terminal domains of these proteins than with the prototypical SH3 domains ( Fig.…”

Stress Physiology of Lactic Acid Bacteria

“…ScaR represses the expression of scaCBA in the presence of Mn 2ϩ . Biochemical studies of the S. gordonii and S. pneumoniae ScaR proteins have shown that ScaR is a homodimer and contains two metal-binding sites per protomer (85,86). In S. pneumoniae ScaR, Zn 2ϩ occupies site 1, and although it is required for activation, it keeps ScaR in an inactive state.…”

Comparative Genomics of DtxR Family Regulons for Metal Homeostasis in Archaea

“…The ferric uptake regulator Fur and zinc uptake regulator Zur constitute the majority of the Fur family metalloregulators, being widely distributed in diverse lineages of Bacteria but not Archaea, whereas the manganese-and nickel-specific regulators Mur and Nur were found in only some bacterial lineages (9,10). Manganese-responsive DtxR family regulators were studied in many bacteria, including MntR in Escherichia coli (11), Bacillus subtilis (12), Staphylococcus aureus (13), Corynebacterium diphtheria (14), and C. glutamicum (15,16), ScaR in Streptococcus gordonii (17), and TroR in Treponema pallidum (18), where they mostly control genes for manganese uptake transporters. In contrast, iron-responsive TFs from the DtxR family were found only in Actinobacteria and include two experimentally studied TFs, the diphtheria toxin repressor DtxR in Corynebacterium diphtheriae (19) and the iron-dependent regulator IdeR in Mycobacterium tuberculosis (20,21), which control large networks of genes involved in iron homeostasis and other cellular functions, such as the toxin gene in C. diphtheriae.…”