Staphylococcus aureusCzrA is a zinc-dependent transcriptional repressor from the ubiquitous ArsR family of metal sensor proteins. Zn(II) binds to a pair of intersubunit C-terminal ␣5-sensing sites, some 15 Å distant from the DNA-binding interface, and allosterically inhibits DNA binding. This regulation is characterized by a large allosteric coupling free energy (⌬Gc) of approximately ؉6 kcal mol ؊1 , the molecular origin of which is poorly understood. Here, we report the solution quaternary structure of homodimeric CzrA bound to a palindromic 28-bp czr operator, a structure that provides an opportunity to compare the two allosteric ''end'' states of an ArsR family sensor. Zn ( M etal ion homeostasis is a complex process that involves maintaining a delicate balance between metal uptake/ efflux and other metal storage systems to meet the needs of the cell (1, 2). This process is tightly regulated at the level of transcription by means of specific metal-dependent transcriptional regulators that respond to changes in metal ion concentrations in the host environment (3). In Staphylococcus aureus, the czr operon encodes a CDF antiporter, CzrB, a homolog of Escherichia coli zinc transporter YiiP that confers resistance to Zn(II) and Co(II) (4, 5), and the metal-regulated repressor, CzrA (6, 7). CzrA binds Zn(II) with picomolar affinity and strong negative homotropic cooperativity (8, 9) and is thought to undergo a conformational change that alleviates transcriptional repression of the resistance gene czrB.CzrA belongs to the ubiquitous ArsR (or ArsR/SmtB) family of metalloregulators found in many bacterial genomes that sense a wide variety of metals including biologically essential metals as well as toxic metal pollutants (10, 11). Members of this family appear to adopt a common winged helix-turn-helix homodimeric fold, but have evolved physically and structurally distinct pairs of allosteric metal-sensing sites (2, 12). These sites are thought to have arisen as a result of convergent evolution due to evolutionary pressures (12), a finding consistent with the ''rule of varied allosteric control'' in which protein families evolve seemingly random allosteric control pathways (13). CzrA and its homolog SmtB in Synechococcus (14) are ␣5 sensors that bind Zn(II) ions in two rotationally symmetric tetrahedral coordination sites formed by pairs of metal ligands derived from the ␣5 helix of each subunit (8). The crystal structure of CzrA and SmtB in the apo and the Zn(II)-bound states have been solved (8). Although the structures of CzrA were found to be very similar in these two states, SmtB revealed measurable differences in quaternary structure in which the apo form adopted a comparatively ''flat'' conformation not well suited to interact with canonical B-form DNA (8,15). The structural and thermodynamic underpinnings of metalloregulation for any member of the ubiquitous ArsR family remains poorly understood due to a lack of detailed insight for the DNA operator-bound state (3).We report here the NMR solution structure of ...
Prokaryotic organisms have evolved an impressive capacity to quickly adapt to a changing and challenging microenvironment in which the availability of both biologically required and non-essential transition metal ions can vary dramatically. In all bacteria, a panel of metalloregulatory proteins control the expression of genes encoding membrane transporters and metal trafficking proteins, that collectively manage metal homeostasis and resistance. These “metal sensors” are specialized allosteric proteins, in which the direct binding of a specific or small number of “cognate” metal ion(s) drives a conformational change in the regulator that allosterically activates or inhibits operator DNA binding, or alternatively, distorts the promoter structure thereby converting a poor promoter to a strong one. In this review, we discuss our current understanding of the features that control metal specificity of the allosteric response in these systems, and the role that structure, thermodynamics and conformational dynamics play in mediating allosteric activation or inhibition of DNA binding.
Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.
Summary CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. We report that CLYBL loss leads to a cell autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.
The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal sensing proteins, and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97’ NHε2•••O=C-His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ΔGc. A “cavity” introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a dramatic loss of regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild-type. The energetics of Zn(II) binding and heterotropic coupling free energies (ΔHc, −TΔSc) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II) sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins results from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression.
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