The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor
The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor
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DOI：
10.1016/j.jmb.2010.08.030
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发表时间：
2010-10-22
影响因子：
5.6
通讯作者：
Giedroc, David P.
中科院分区：
文献类型：
--
作者：
Reyes-Caballero, Hermes;Guerra, Alfredo J.;Giedroc, David P.
肺炎链球菌D39AdcR(粘附素能力抑制因子)是MARR(多重抗生素耐药抑制因子)家族中第一个被鉴定的金属敏感成员。在微氧条件下用液体培养(脑心输液)培养的Delta adcR菌株的表达谱显示，13个基因上调，包括编码对锌的高亲和力ABC摄取系统的adcR和adcCBA，以及编码细胞表面锌结合的肺炎球菌组氨酸三联体(PHT)蛋白和AdcAII(LMB，层粘连蛋白结合)的基因。在0.2 mM锌(II)中生长的Delta adcR、H108Q和H112Q adcR突变等位基因菌株表现出生长缓慢的表型，细胞相关锌(II)的含量增加了近两倍。Apo和Zn(II)结合的AdcR在溶液中是同源二聚体，与含有ADC操纵子的28聚体DNA的结合可被KDNA-Zn=2.4×10(8)M-1(pH 6.0，0.2M氯化钠，25℃)的锌(II)强烈刺激。AdcR每二聚体结合两个锌(II)，在pH 6.0和pH 8.0时，>；=10(9)M-1和>；=10(12)M-1的锌(II)亲和力分别为K-Zn1和K-Zn2，根据pH的不同有一到三个低亲和力的锌(II)。高亲和力部位的X射线吸收光谱显示了一个五配位的N/O络合物，没有半胱氨酸配位，后者的发现得到了C30A AdcR野生型功能性质的证实。丙氨酸取代DNA结合区的保守残基His42和C末端调节区的His108和His112，取消了高亲和力锌(L)的结合，大大减少了锌(II)激活的与DNA的结合。核磁共振研究表明，这些突变体采用与二聚体野生型apo-AdcR相同的折叠构象，但不能与锌(II)结合进行构象转换。这些研究表明，His42、His108和H112是肺炎链球菌AdcR中的金属调节锌配体。(C)2010爱思唯尔有限公司。保留所有权利。
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 Delta 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 Delta 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 x 10(8) M-1 (pH 6.0, 0.2 M NaCl, 25 degrees 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(l) 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. (C) 2010 Elsevier Ltd. All rights reserved.