Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition
Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition
复制标题
DOI：
10.1110/ps.062425706
复制
发表时间：
2006-11-01
期刊：
影响因子：
8
通讯作者：
Tanner, John J.
中科院分区：
文献类型：
--
作者：
Larson, John D.;Jenkins, Jermaine L.;Tanner, John J.
PutA（proline utilization A，脯氨酸利用A）是大肠杆菌的一个含1320个氨基酸残基的蛋白质，它既是一种双功能脯氨酸分解代谢酶，又是一种内源性转录抑制因子。在这里，我们报告的第一个晶体结构的PutA DNA结合域沿着与功能分析的突变PutA缺陷的DNA结合。使用对应于大肠杆菌残基1-52的多肽生长晶体。coli PutA（PutA52）。使用Se-Met MAD定相确定PutA 52突变体Lys 9 Met的2.1埃分辨率结构，并且使用分子置换以1.9埃分辨率解析天然PutA 52的结构。残基3-46形成带-螺旋-螺旋（RHH）亚结构，从而确立PutA为含有RHH结构域的最大蛋白质。PutA RHH结构域形成具有紧密堆积的疏水性核心的相互交织的二聚体，这是RHH家族的特征。这些结构用于检查PutA RHH结构域中保守残基的三维背景。同源性建模表明，Lys 9和Thr 5通过大沟接触DNA碱基，而Arg 15、Thr 28和His 30可能与磷酸骨架相互作用。Lys 9被证明是必不可少的特异性识别的投入控制DNA使用的Lys 9 Met突变体的全长PutA的凝胶位移分析。Lys 9在PutA 52结构中是无序的，这意味着一种诱导配合结合机制，其中Lys 9的侧链通过与DNA的相互作用而变得有序。这些结果提供了新的见解的DNA识别的结构基础PutA和揭示PutA二聚体界面的三维结构细节。
PutA (proline utilization A) from Escherichia coli is a 1320-amino-acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1-52 of E. coli PutA (PutA52). The 2.1 angstrom resolution structure of PutA52 mutant Lys9Met was determined using Se-Met MAD phasing, and the structure of native PutA52 was solved at 1.9 angstrom resolution using molecular replacement. Residues 3-46 form a ribbon-helix-helix (RHH) substructure, thus establishing PutA as the largest protein to contain an RHH domain. The PutA RHH domain forms the intertwined dimer with tightly packed hydrophobic core that is characteristic of the RHH family. The structures were used to examine the three-dimensional context of residues conserved in PutA RHH domains. Homology modeling suggests that Lys9 and Thr5 contact DNA bases through the major groove, while Arg15, Thr28, and His30 may interact with the phosphate backbone. Lys9 is shown to be essential for specific recognition of put control DNA using gel shift analysis of the Lys9Met mutant of full-length PutA. Lys9 is disordered in the PutA52 structure, which implies an induced-fit binding mechanism in which the side chain of Lys9 becomes ordered through interaction with DNA. These results provide new insights into the structural basis of DNA recognition by PutA and reveal three-dimensional structural details of the PutA dimer interface.