Heme binding to cold shock protein D, CspD, from Vibrio cholerae
Heme binding to cold shock protein D, CspD, from Vibrio cholerae
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血红素与霍乱弧菌冷休克蛋白 D、CspD 结合
DOI：
10.1016/j.bbrc.2022.07.074
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发表时间：
2022
影响因子：
3.1
通讯作者：
Uchida Takeshi
中科院分区：
文献类型：
--
作者：
Nam Dayeon;Motegi Wataru;Ishimori Koichiro;Uchida Takeshi
冷休克蛋白D（CspD）是冷休克蛋白A（CspA）的同源蛋白之一，通过与单链DNA结合来抑制DNA复制。我们发现霍乱弧菌的CspD（VcCspD）具有一个血红素调节基序（HRM）序列，并以1：1的化学计量比特异性结合血红素。合成的单链DNA寡聚体（ssDNA）的结合之后是Trp的荧光猝灭。在血红素存在下，与ssDNA的加入相关的荧光猝灭被抑制，表明血红素与VcCspD的结合抑制了VcCspD-ssDNA复合物的形成。对于用丙氨酸（C22 A）替换HRM中的Cys 22的VcCspD突变体，没有观察到这种血红素诱导的抑制。因此，在Cys 22处的血红素结合对于抑制ssDNA与VcCspD的结合是必需的。当过量表达VcCspD时，大肠杆菌在37 °C下的生长减慢，表明VcCspD阻碍大肠杆菌的生长。当通过添加血红素前体δ-氨基乙酰丙酸促进细胞中血红素的产生时，表达VcCspD的大肠杆菌的生长减慢，但表达C22 A突变体的大肠杆菌的生长没有减慢。这些观察结果使我们能够得出结论，血红素特异性地结合到HRM区域inVcCspD和抑制目标ssDNA的结合，这表明血红素作为DNA复制的调节分子发挥作用。
Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding toVcCspD inhibited the formation of theVcCspD-ssDNA complex. Such heme-induced inhibition was not observed for theVcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding forVcCspD. The growth ofEscherichia coliat 37 °C was slowed whenVcCspD was overexpressed, indicating thatVcCspD hampers the growth ofE. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth ofE. coliexpressingVcCspD was decelerated, but the growth ofE. coliexpressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region inVcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.