The Metalloregulatory Zinc Site in Streptococcus Pneumoniae AdcR, a Zinc-Activated MarR Family Repressor

J Mol Biol. 2010 Oct 22;403(2):197-216. doi: 10.1016/j.jmb.2010.08.030. Epub 2010 Sep 8.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • DNA, Bacterial / metabolism
  • Gene Deletion
  • Gene Expression Profiling
  • Gene Expression Regulation, Bacterial
  • Kinetics
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Multimerization
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Streptococcus pneumoniae / chemistry
  • Streptococcus pneumoniae / metabolism*
  • X-Ray Absorption Spectroscopy
  • Zinc / metabolism*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Repressor Proteins
  • Zinc