Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.
Characterization of the Catalytic Domain of Human APOBEC3B and the Critical Structural Role for a Conserved Methionine.
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人 APOBEC3B 催化结构域的表征以及保守蛋氨酸的关键结构作用。
DOI：
10.1016/j.jmb.2015.08.006
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发表时间：
2015
影响因子：
5.6
通讯作者：
Bhagwat,AshokS
中科院分区：
文献类型：
--
作者：
Siriwardena,SachiniU;Guruge,ThisariA;Bhagwat,AshokS
人APOBEC 3B使DNA中的胞嘧啶脱氨基，并且属于酶的AID/APOBEC家族。这些蛋白质参与先天性和适应性免疫，并可能导致各种癌症的突变。为了表征其将胞嘧啶转化为尿嘧啶的能力，我们测试了APOBEC 3B基因的几种衍生物在大肠杆菌中引起突变的能力。通过该分析，发现氨基末端结构域（NTD）和羧基末端结构域（CTD）连接处的甲硫氨酸残基对高致突变性至关重要。在该位置具有取代的突变体的性质、对APOBEC 3家族成员的现有分子结构的检查和分子建模表明，该残基对于该蛋白家族的结构稳定性是必不可少的。将具有最高突变活性的APOBEC 3B CTD纯化至均一，并测定其动力学参数。CTD单体的尺寸排阻色谱法表明，它与其二聚体形式处于平衡状态，并且蛋白质的基质辅助激光解吸电离飞行时间分析表明，二聚体可能相当稳定。部分纯化的NTD没有表现出内在的脱氨活性，并没有提高CTD在生化测定中的活性。最后，在遗传测定中，APOBEC 3B将5-甲基胞嘧啶（5 mC）突变为胸腺嘧啶的效率比APOBEC 3A低至少10倍，并且在生物化学测定中，与C相比，APOBEC 3B将5 mC脱氨的效率低至少10倍。这些结果揭示了APOBEC 3B催化结构域的结构组织，其底物特异性及其在引起全基因组突变中的可能作用。
Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines into uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations inEscherichia coli. Through this analysis, a methionine residue at the junction of the amino-terminal domain (NTD) and the carboxy-terminal domain (CTD) was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B CTD with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size-exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and matrix-assisted laser desorption ionization time-of-flight analysis of the protein suggested that the dimer may be quite stable. The partially purified NTD did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations.