Department of Chemistry and ‡Interdisciplinary Graduate Program in Biochemistry, Indiana University , Bloomington, Indiana 47405-7102, United States.
Biochemistry. 2013 Oct 29;52(43):7689-701. doi: 10.1021/bi401132w. Epub 2013 Oct 14.
Transition metals, including manganese, are required for the proper virulence and persistence of many pathogenic bacteria. In Streptococcus pneumoniae (Spn), manganese homeostasis is controlled by a high-affinity Mn(II) uptake complex, PsaBCA, and a constitutively expressed efflux transporter, MntE. psaBCA expression is transcriptionally regulated by the DtxR/MntR family metalloregulatory protein pneumococcal surface antigen repressor (PsaR) in Spn. Here, we present a comprehensive analysis of the metal and DNA binding properties of PsaR. PsaR is a homodimer in the absence and presence of metals and binds two manganese or zinc atoms per protomer (four per dimer) in two pairs of structurally distinct sites, termed site 1 and site 2. Site 1 is likely filled with Zn(II) in vivo (K(Zn1) ≥ 10¹³ M⁻¹; K(Mn1) ≈ 10⁸ M⁻¹). The Zn(II)-site 1 complex adopts a pentacoordinate geometry as determined by X-ray absorption spectroscopy containing a single cysteine and appears to be analogous to the Cd(II) site observed in Streptococcus gordonii ScaR. Site 1 is necessary but not sufficient for full positive allosteric activation of DNA operator binding by metals as measured by ΔGc, the allosteric coupling free energy, because site 1 mutants show an intermediate ΔGc. Site 2 is the primary regulatory site and governs specificity for Mn(II) over Zn(II) in PsaR, where ΔGc(Zn,Mn) >> ΔGc(Zn,Zn) despite the fact that Zn(II) binds site 2 with an affinity 40-fold higher than that of Mn(II); i.e., K(Zn2) > K(Mn2). Mutational studies reveal that Asp7 in site 2 is a critical ligand for Mn(II)-dependent allosteric activation of DNA binding. These findings are discussed in the context of other well-studied DtxR/MntR Mn(II)/Fe(II) metallorepressors.
过渡金属，包括锰，是许多致病性细菌适当毒力和持续存在所必需的。在肺炎链球菌（Spn）中，锰稳态由高亲和力 Mn（II）摄取复合物 PsaBCA 和组成型表达的外排转运蛋白 MntE 控制。PsaBCA 的表达受 Spn 中 DtxR/MntR 家族金属调节蛋白肺炎球菌表面抗原阻遏物（PsaR）的转录调控。在这里，我们对 PsaR 的金属和 DNA 结合特性进行了全面分析。PsaR 在没有和存在金属的情况下是同源二聚体，每个亚基（二聚体中每个）结合两个锰或锌原子，在两个结构不同的位点（称为位点 1 和位点 2）结合两对。位点 1 可能在体内充满 Zn（II）（K（Zn1）≥10¹³ M⁻¹；K（Mn1）≈10⁸ M⁻¹）。X 射线吸收光谱法确定 Zn（II）-位点 1 配合物采用五配位几何形状，包含一个单一的半胱氨酸，并且似乎类似于在链球菌戈登氏菌 ScaR 中观察到的 Cd（II）位点。位点 1 对于金属对 DNA 操作子结合的完全正变构激活是必需的，但不是充分的，如通过 ΔGc 测量的，变构偶联自由能，因为位点 1 突变体显示出中间的 ΔGc。位点 2 是主要调节位点，控制 PsaR 中 Mn（II）与 Zn（II）的特异性，尽管 Zn（II）与位点 2 的结合亲和力比 Mn（II）高 40 倍，但 ΔGc（Zn，Mn）>> ΔGc（Zn，Zn）；即，K（Zn2）> K（Mn2）。突变研究表明，位点 2 中的天冬氨酸 7 是 Mn（II）依赖的 DNA 结合变构激活的关键配体。这些发现是在其他研究充分的 DtxR/MntR Mn（II）/Fe（II）金属阻遏物的背景下讨论的。
