Structural and mutational analyses of Aes, an inhibitor of MalT in Escherichia coli
Structural and mutational analyses of Aes, an inhibitor of MalT in Escherichia coli
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DOI：
10.1002/prot.24383
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发表时间：
2014-02
期刊：
影响因子：
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通讯作者：
A. Schiefner;K. Gerber;A. Brosig;W. Boos
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文献类型：
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作者：
A. Schiefner;K. Gerber;A. Brosig;W. Boos
在大肠杆菌中，酰基酯酶Aes利用基因有效地抑制了麦芽糖和麦芽糖糊精的中心激活剂麦芽的转录活性。为了更好地了解Aes和MalT之间相互作用的性质，我们确定了Aes的两种不同的晶体结构——一种是天然形式，另一种是在活性位点丝氨酸上被苯基甲基磺酰基部分共价修饰。这两种结构显示出不同的空间群，分别被细化到1.8 Å和2.3 Å的分辨率。Aes的整体结构类似于典型的α/β水解酶折叠，它由漏斗状帽结构延伸，形成底物结合位点。由Ser165、His292和Asp262组成的催化三元组位于漏斗的底部。通过分析两种不同空间群的晶体堆积接触以及分析尺寸-排斥色谱，发现Aes是同二聚体排列。Aes二聚体采用涉及水解酶核和帽的反平行接触，其双轴垂直于Aes的最大尺寸。为了确定Aes与MalT相互作用的表面积，我们进行了基于结构的丙氨酸扫描诱变，以确定在MalT抑制中显着受损的Aes残基，但仍然表现出野生型表达和酶活性。这些残基映射到二聚化界面相反位置的Aes的浅微凹表面斑块，并指示与MalT相互作用的表面积。蛋白质2014;82:268 - 277。©2013 Wiley期刊公司
The acyl esterase Aes effectively inhibits the transcriptional activity of MalT—the central activator of maltose and maltodextrin utilizing genes in Escherichia coli. To provide better insight into the nature of the interaction between Aes and MalT, we determined two different crystal structures of Aes—in its native form and covalently modified by a phenylmethylsulfonyl moiety at its active site serine. Both structures show distinct space groups and were refined to a resolution of 1.8 Å and 2.3 Å, respectively. The overall structure of Aes resembles a canonical α/β‐hydrolase fold, which is extended by a funnel‐like cap structure that forms the substrate‐binding site. The catalytic triad of Aes, comprising residues Ser165, His292, and Asp262, is located at the bottom of this funnel. Analysis of the crystal‐packing contacts of the two different space groups as well as analytical size‐exclusion chromatography revealed a homodimeric arrangement of Aes. The Aes dimer adopts an antiparallel contact involving both the hydrolase core and the cap, with its twofold axis perpendicular to the largest dimension of Aes. To identify the surface area of Aes that is responsible for the interaction with MalT, we performed a structure‐based alanine‐scanning mutagenesis to pinpoint Aes residues that are significantly impaired in MalT inhibition, but still exhibit wild‐type expression and enzymatic activity. These residues map to a shallow slightly concave surface patch of Aes at the opposite site of the dimerization interface and indicate the surface area that interacts with MalT. Proteins 2014; 82:268–277. © 2013 Wiley Periodicals, Inc.