Sharadamma Narayanaswamy, Harshavardhana Yadumurthy, Ravishankar Apoorva, Anand Praveen, Chandra Nagasuma, Muniyappa K
From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
J Biol Chem. 2014 Dec 5;289(49):34325-40. doi: 10.1074/jbc.M114.608596. Epub 2014 Oct 16.
The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.
结核分枝杆菌的全基因组注释序列显示，Rv1388（Mtihf）可能编码一种假定的20 kDa整合宿主因子（mIHF）。然而，关于mIHF的功能特性或分枝杆菌类核的组织情况，人们知之甚少。基于天蓝色链霉菌IHF双链DNA（mIHF的直系同源物）的共晶体结构对mIHF三维结构进行分子建模，发现存在可能参与DNA结合的Arg-170、Arg-171和Arg-173，以及在紧密转角处的一个保守脯氨酸（Pro-150）。大肠杆菌ΔihfA和ΔihfB菌株对紫外线和甲磺酸甲酯的表型敏感性可以被野生型Mtihf互补，但不能被其在DNA结合残基上携带突变的等位基因互补。蛋白质-DNA相互作用分析表明，野生型mIHF而非其DNA结合变体，与含有attB和attP位点以及弯曲DNA的片段具有高亲和力结合。令人惊讶的是，mIHF在功能上重要的氨基酸残基及其与DNA结合、DNA弯曲和位点特异性重组的潜在机制与大肠杆菌IHFαβ的根本不同。此外，我们揭示了关于IHF介导的DNA压缩的新见解，这取决于其偏好结合位点的位置；mIHF促进DNA压缩成类核状或更高阶的丝状结构。因此我们提出，mIHF是一类蛋白质亚家族的独特成员，这些蛋白质在位点特异性重组和类核组织中作为必需的辅助因子，并且这些发现代表了我们对类核相关蛋白作用的理解有了重大进展。
