Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.
Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.
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DOI：
10.1016/j.jmb.2015.07.014
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发表时间：
2015-08-28
影响因子：
5.6
通讯作者：
Kleanthous C
中科院分区：
文献类型：
--
作者：
Joshi A;Grinter R;Josts I;Chen S;Wojdyla JA;Lowe ED;Kaminska R;Sharp C;McCaughey L;Roszak AW;Cogdell RJ;Byron O;Walker D;Kleanthous C
超高亲和力蛋白质-蛋白质相互作用如何保持高特异性仍然知之甚少。粘菌素DNase结构域与其抑制免疫(Im)蛋白之间的相互作用是一种超高亲和力的相互作用，对于中和内源性DNase催化活性和保护外源DNase细菌素是必不可少的。粘菌素DNase-Im相互作用是研究高亲和力蛋白质-蛋白质相互作用的模型系统。然而，尽管革兰氏阴性细菌广泛产生与粘连素样细菌素密切相关的细菌素，但这种相互作用只被用来自大肠杆菌的粘菌素来研究。在本工作中，我们首次报道了铜绿假单胞菌DNase-Im复合体的晶体结构，它们分别是Pocin S2 DNase-ImS2和Pocin AP41 DNase-ImAP41。这些结构代表了不同的DNase-Im亚家族，对于扩大我们对这类重要的高亲和力蛋白质复合体的蛋白质-蛋白质相互作用的理解具有重要意义。这项工作的一个关键发现是，免疫蛋白结合能量热点Helix III中的突变可以被DNA酶与免疫蛋白结合界面上的互补替换所容忍。IM螺旋III在粘连蛋白中是严格保守的，其中天冬氨酸与DNA酶骨架形成极性相互作用。ImAP41在螺旋III中含有天冬氨酸到甘氨酸的取代，我们的结构表明了共同进化取代的作用，其中DNase环4中的Pro占据了腾出的体积，并消除了未完成的氢键。我们观察到其他DNA酶免疫对中的共同进化突变，这似乎是该家族分裂为两个不同群体的基础。我们已经鉴定了两个不同的细菌素DNase-Im亚家族。与中和Im蛋白形成的复合体中的腐蛋白脱氧核糖核酸酶结构。这些亚家族的特征是具有不同的Im螺旋III基序。ImAP41缺乏Im Helix III的关键天冬氨酸，也是一种保守的界面水。新的DNase-Im家族扩大了控制细菌素选择性的区域。
How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. We have identified two different bacteriocin DNase–Im subfamilies. First structures of pyocin DNase domains in complex with neutralising Im proteins. The subfamilies are characterised by distinct Im helix III motifs. ImAP41 lacks the key Asp in Im helix III and one of the conserved interfacial waters. New DNase–Im family expands the region that governs bacteriocin selectivity.