Mutation of leucine 20 causes a change of local conformation indirectly impairing the DNA binding of SP_0782 from Streptococcus pneumoniae
Mutation of leucine 20 causes a change of local conformation indirectly impairing the DNA binding of SP_0782 from Streptococcus pneumoniae
复制标题
亮氨酸 20 突变导致局部构象变化，间接损害肺炎链球菌 SP_0782 的 DNA 结合
DOI：
10.1016/j.bbrc.2020.01.057
复制
发表时间：
2020-03-26
影响因子：
3.1
通讯作者：
Liu, Maili
中科院分区：
文献类型：
--
作者：
Gong, Yixuan;Li, Shuangli;Liu, Maili
肺炎链球菌SP_0782是一种二聚体PC4样蛋白结合单链DNA(SsDNA)，可能参与维持基因组的稳定和自然转化。SP_0782通过在其两个DNA结合区(DBRs)上不同地容纳核苷酸，以不同的方式与不同长度的单链DNA结合。在这里，我们报告了一个新的位点，亮氨酸20(L20)，它不位于DBRS中，但当突变为丙氨酸(L20A)时会损害DNA结合。L20A突变显著降低了SP_0782与单链DNA dT19G1的DNA结合亲和力，并影响了SP_0782：dT19G1高阶复合体的形成。L20的侧链与apo SP0782结构中DBRS的几个残基发生相互作用，L20A突变导致圆二色谱(CD)和核磁共振谱宽化学位移微扰(CSPs)的变化。核磁共振谱上受影响最大的残基包括位于DBR2的F39和R49，以及位于DBR1的K60，这可能是SP_0782二聚体中两个亚基协同结合单链DNA的重要原因。因此，L20A突变导致SP_0782的局部构象改变，间接影响DNA结合界面，从而削弱与单链DNA dT19G1的亲和力。有趣的是，这个L20位点在细菌中是保守的，但在真核细胞中不是PC4样蛋白，这表明进化上的分歧。本研究揭示了SP_0782的结构与功能的关系，以及可能具有选择性抑菌作用的氨基酸位点。(C)2020 Elsevier Inc.保留所有权利。
SP_0782 from Streptococcus pneumoniae is a dimeric PC4-like protein binding single-stranded DNA (ssDNA), and is potentially involved in maintenance of genome stability and natural transformation. SP_0782 binds with different lengths of ssDNA in various patterns through accommodating nucleotides differently in its two DNA-binding regions (DBRs). Here, we report the characterization of a novel site, leucine 20 (L20), which is not located in the DBRs but impairs the DNA binding when mutated to alanine (L20A). The L20A mutation markedly reduced the DNA-binding affinity of SP_0782 for ssDNA dT19G1, and affected the formation of high-order SP_0782:dT19G1 complexes. The side chain of L20 shows interactions with several residues at the backside of the DBRs in apo SP_0782 structure, and the L20A mutation led to a change of circular dichroism (CD) spectrum and broad chemical shift perturbations (CSPs) in NMR spectrum compared with the wild type. The most affected residues in NMR spectrum included F39 and R49 located in DBR2, as well as K60 in DBR1, which was suggested to be important for cooperative binding of ssDNA by the two subunits in SP_0782 dimer. Thus, the L20A mutation caused a local conformational change of SP_0782, which exerted an indirect effect on the DNA-binding interface and therefore impaired the affinity for ssDNA dT19G1. Interestingly, this L20 site is conserved in bacterial but not eukaryotic PC4-like proteins, suggesting an evolutionary divergence. This study provides an insight into the structure-function relationship of SP_0782, and an amino-acid site probably targeted for inhibiting bacteria selectively. (C) 2020 Elsevier Inc. All rights reserved.