Opening of a cryptic pocket in β-lactamase increases penicillinase activity.
Opening of a cryptic pocket in β-lactamase increases penicillinase activity.
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DOI：
10.1073/pnas.2106473118
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发表时间：
2021-11-23
影响因子：
11.1
通讯作者：
Bowman GR
中科院分区：
文献类型：
--
作者：
Knoverek CR;Mallimadugula UL;Singh S;Rennella E;Frederick TE;Yuwen T;Raavicharla S;Kay LE;Bowman GR
蛋白质是一种变形者，但目前还不清楚蛋白质可以采用的许多结构中哪些与其功能相关。在这里，我们检查含有“隐蔽”口袋的构象（即，无配体结构中不存在的口袋）。隐藏口袋在药物发现工作中具有潜在的效用，因为它们提供了靶向“不可药物化的”蛋白质（即，缺乏已知口袋的蛋白质）或增强而不是抑制蛋白质功能。在这项研究中，我们使用巯基标记和动力学分析，NMR和分子动力学模拟的组合，以确定在β-内酰胺酶的Ω-环隐蔽口袋的功能。我们发现，一个开放的口袋人口是有益的水解底物苄青霉素。了解蛋白质激发态的功能作用对蛋白质设计和药物发现具有重要意义。然而，由于这些状态很难发现和研究，目前还不清楚激发态是否仅仅是由热波动引起的，通常会减损功能，或者这些状态是否真的可以增强蛋白质功能。为了研究这个问题，我们考虑了β-内酰胺酶中的激发态，特别是含有在Ω环下形成的隐蔽口袋的状态的子集。鉴于Ω环的已知重要性以及至少两种同系物中存在该口袋，我们假设这些激发态增强酶活性。使用巯基标记测定来探测Ω-环口袋动力学和动力学测定来探测活性，我们发现虽然该口袋在β-内酰胺酶同系物中不完全保守，但具有Ω-环口袋的那些对底物苄青霉素具有更高的活性。我们还发现，这对于具有更大的开环Ω-环口袋群体的TEM β-内酰胺酶变体是正确的。我们进一步研究开放人口使用NMR化学交换饱和转移实验和分子动力学模拟相结合。为了测试我们对Ω环口袋的功能作用的理解，我们设计了突变以增强/抑制口袋打开，并观察到苄青霉素活性与我们设计的变体中口袋打开的概率成比例。这里描述的工作表明，含有隐蔽口袋的激发态可能有利于功能，并且可能受到自然选择的青睐，从而增加了这种隐蔽口袋作为药物靶标的潜在效用。
A protein is a shape-shifter, but it is currently unclear which of the many structures a protein can adopt are relevant for its function. Here, we examine conformations that contain a “cryptic” pocket (i.e., a pocket absent in ligand-free structures). Cryptic pockets have potential utility in drug discovery efforts because they provide a means to target “undruggable” proteins (i.e., proteins lacking known pockets) or enhance rather than inhibit protein function. In this study, we use a combination of thiol-labeling and kinetic assays, NMR, and molecular dynamic simulations to identify the function of the Ω-loop cryptic pocket in β-lactamase enzymes. We find that an open pocket population is beneficial for hydrolysis of the substrate benzylpenicillin. Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.
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DOI：
10.1073/pnas.2024725118
发表时间：
2021-03-16
影响因子：
11.1
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通讯作者：
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