title: The metalloregulatory zinc site in Streptococcus pneumoniae AdcR, a zinc-activated MarR family repressor.
abstract: Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K(DNA-Zn)=2.4 × 10(8) M(-1) (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K(Zn1) and K(Zn2) of ≥10(9) M(-1) at pH 6.0 and ≥10(12) M(-1) at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, USA.
authors.forename: Hermes
authors.initials: H
authors.lastname: Reyes-Caballero
authors.forename: Alfredo J
authors.initials: AJ
authors.lastname: Guerra
authors.forename: Faith E
authors.initials: FE
authors.lastname: Jacobsen
authors.forename: Krystyna M
authors.initials: KM
authors.lastname: Kazmierczak
authors.forename: Darin
authors.initials: D
authors.lastname: Cowart
authors.forename: Uma Mahendra Kumar
authors.initials: UM
authors.lastname: Koppolu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Scott
authors.forename: Malcolm E
authors.initials: ME
authors.lastname: Winkler
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D055503:Protein Multimerization; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000378:metabolism*; D056928:X-Ray Absorption Spectroscopy; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D015032:Zinc
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