TY - JOUR
T1 - The metalloregulatory zinc site in streptococcus pneumoniae AdcR, a Zinc-activated MarR family repressor
AU - Reyes-Caballero, Hermes
AU - Guerra, Alfredo J.
AU - Jacobsen, Faith E.
AU - Kazmierczak, Krystyna M.
AU - Cowart, Darin
AU - Koppolu, Uma Mahendra Kumar
AU - Scott, Robert A.
AU - Winkler, Malcolm E.
AU - Giedroc, David P.
N1 - Funding Information:
We thank Yue Fu for his help in protein purification and Dhriti Mukherjee and Kyle J. Wayne for their assistance with S. pneumoniae D39 strain construction. We also wish to thank Drs. Jon Karty, Todd Stone and Randy Arnold of Indiana University for training and technical support during various stages of this work. This work was supported by grants from the NIH GM042569 (to D.P.G.), F32 AI084445 (to F.E.J.), GM042025 (to R.A.S.) and AI060744 (to M.E.W).
PY - 2010/10/22
Y1 - 2010/10/22
N2 - 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 {increment}adcR 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 {increment}adcR, 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 KDNA-Zn=2.4×108M-1 (pH 6.0, 0.2M NaCl, 25°C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities KZn1 and KZn2 of ≥109M-1 at pH 6.0 and ≥1012M-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.
AB - 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 {increment}adcR 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 {increment}adcR, 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 KDNA-Zn=2.4×108M-1 (pH 6.0, 0.2M NaCl, 25°C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities KZn1 and KZn2 of ≥109M-1 at pH 6.0 and ≥1012M-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.
KW - Bacterial pathogen
KW - Metalloregulation
KW - Zinc homeostasis
KW - Zinc sensor
KW - Zinc-activated repressor
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U2 - 10.1016/j.jmb.2010.08.030
DO - 10.1016/j.jmb.2010.08.030
M3 - Article
C2 - 20804771
AN - SCOPUS:77957233008
SN - 0022-2836
VL - 403
SP - 197
EP - 216
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 2
ER -