Physical Characterization of the Manganese-Sensing Pneumococcal Surface Antigen Repressor from Streptococcus pneumoniae

详细信息    查看全文

• 作者：John P. Lisher ; Khadine A. Higgins ; Michael J. Maroney ; David P. Giedroc
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：October 29, 2013
• 年：2013
• 卷：52
• 期：43
• 页码：7689-7701
• 全文大小：533K
• 年卷期：v.52,no.43(October 29, 2013)
• ISSN：1520-4995

文摘

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 鈮?10¹³ M^鈥?; K_Mn1 鈮?10⁸ M^鈥?). The Zn(II)鈥搒ite 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 螖G_c, the allosteric coupling free energy, because site 1 mutants show an intermediate 螖G_c. Site 2 is the primary regulatory site and governs specificity for Mn(II) over Zn(II) in PsaR, where 螖G_c^Zn,Mn 螖G_c^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.