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Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.

Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, P(m), requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a -10 hexamer, but no -35 hexamer. Instead P(m) has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing -35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a β-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the β-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the β-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A(3) demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.

Transcription from the middle promoter, Pm, of bacteriophage Mu requires the phage-encoded activator protein Mor and bacterial RNA polymerase. Mor is a sequence-specific DNA-binding protein that mediates transcription activation through its interactions with the C-terminal domains of the alpha and sigma subunits of bacterial RNA polymerase. Here we present the first structure for a member of the Mor/C family of transcription activators, the crystal structure of Mor to 2.2-A resolution. Each monomer of the Mor dimer is composed of two domains, the N-terminal dimerization domain and C-terminal DNA-binding domain, which are connected by a linker containing a beta strand. The N-terminal dimerization domain has an unusual mode of dimerization; helices alpha1 and alpha2 of both monomers are intertwined to form a four-helix bundle, generating a hydrophobic core that is further stabilized by antiparallel interactions between the two beta strands. Mutational analysis of key leucine residues in helix alpha1 demonstrated a role for this hydrophobic core in protein solubility and function. The C-terminal domain has a classical helix-turn-helix DNA-binding motif that is located at opposite ends of the elongated dimer. Since the distance between the two helix-turn-helix motifs is too great to allow binding to two adjacent major grooves of the 16-bp Mor-binding site, we propose that conformational changes in the protein and DNA will be required for Mor to interact with the DNA. The highly conserved glycines flanking the beta strand may act as pivot points, facilitating the conformational changes of Mor, and the DNA may be bent.