Distinguishing direct binding interactions fromallosteric effects in the protease–HK97 prohead Iδ domain complex by amide H/D exchange massspectrometry

S Krishnamurthy; D Veesler; R KhayatJ SnijdercdRk HuangAJR HeckcdJe JohnsonGS Anand

摘要

A major question in mapping protein-ligand or protein-protein interactions in solution is to distinguish direct-binding interactions from long-range conformational changes at allosteric sites. We describe here the applicability of amide hydrogen deuterium exchange mass spectrometry (HDXMS) in addressing this important question using the bacteriophage HK97 capsid proteins’ interactions with their processing protease. HK97 is a lambda-like dsDNA bacteriophage that is ideal for studies of particle assembly and maturation. Its capsid precursor protein is composed of two main regions, the scaffolding protein (δ-domain, residues 2-103), and the coat subunit (residues 104-385), which spontaneously forms a mixture of hexamers and pentamers upon association. Activation of the viral protease, which occurs after particle assembly, is initiated by the protease mediated digestion of the scaffolding domains to yield Prohead-2. This irreversible step is obligatory for activation of the virus maturation pathway.Here we provide an “addendum” to our previous study of Prohead I and Prohead I+pro (a transient complex of Prohead I and the protease) where we investigated the interactions between the δ domain and the packaged protease using HDXMS. Our results revealed two sites on the δ domain: one set of contiguous peptides that showed decreased exchange at the protease binding site at early time points of deuterium labeling and another separate set of continuous peptides that showed decreased exchange at latertime points. Even though this cannot reveal the time scales of molecular processes governing binding and allostery, we believe this differential pattern of exchange across deuteration times can allow spatial distinction between binding sites and long range conformational changes with allosteric implications. This partitioning can be discerned from the lag between noncontiguous regions on a protein showing maximal changes in deuterium exchange and highlights a powerful application of HDXMS in distinguishing direct binding in transient protein-protein interactions from allosteric changes.

机译：在映射protein-ligand或一个主要问题蛋白质-蛋白质之间的关系解决方案是区分direct-binding交互远程变构构象的变化网站。酰胺氢氘交换质量在解决这一重要的光谱法(HDXMS)使用噬菌体HK97衣壳问题蛋白质的交互处理蛋白酶。噬菌体是理想的研究粒子组装和成熟。前体蛋白是由两个主要组成的地区,支架蛋白(δ域,残留2 - 103)和外套亚基(残留物104 - 385年),自发形成的混合物五个一,对协会五聚物。病毒蛋白酶的激活,发生粒子组装后,发起的脚手架的蛋白酶介导的消化域Prohead-2屈服。一步是专为激活的病毒成熟的通路。我们先前的研究Prohead我和Prohead我+专业(Prohead我和瞬变复杂蛋白酶),我们调查了交互δ域和包装之间的蛋白酶使用HDXMS。δ域:一组相邻肽在蛋白酶绑定显示减少交换网站在早期氘标记的时间点和另一组不同的连续的肽在latertime显示减少交换点。尺度的分子过程管理绑定变构效应,我们相信这个微分在含重氢倍可以交换模式让空间结合位点之间的区别和远程构象变化变构的影响。从非连续之间的滞后区域上的蛋白质表现出最大的变化氘交换和凸显了强大HDXMS区分直接的应用绑定在短暂的蛋白质相互作用从变构的变化。

Distinguishing direct binding interactions fromallosteric effects in the protease–HK97 prohead Iδ domain complex by amide H/D exchange massspectrometry

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