A force-activated trip switch triggers rapid dissociation of a colicin from its immunity protein.
A force-activated trip switch triggers rapid dissociation of a colicin from its immunity protein.
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DOI：
10.1371/journal.pbio.1001489
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发表时间：
2013
期刊：
影响因子：
9.8
通讯作者：
Brockwell DJ
中科院分区：
文献类型：
--
作者：
Farrance OE;Hann E;Kaminska R;Housden NG;Derrington SR;Kleanthous C;Radford SE;Brockwell DJ
一项单分子力研究表明，高亲和蛋白相互作用的快速解离可以通过一个蛋白伴侣的位点特异性重塑来触发，并且重塑的预防维持了亲和性。大肠杆菌素是由大肠杆菌菌株合成的蛋白质抗生素，用于靶向和杀死相关细菌。为了防止宿主自杀，粘菌素通过与免疫蛋白结合而失活。尽管免疫蛋白的释放频率很高（Kd≈fM，寿命≈4天），但免疫蛋白的释放是大肠杆菌素中毒的先决条件，中毒发生在几分钟的时间尺度上。在这里，通过使用原子力显微镜测量大肠杆菌素E9 （E9）和免疫蛋白9 （Im9）复合物的dna酶结构域解离的动态力谱，我们发现低力（<20 pN）的应用使复合物的解离率增加了106倍，达到与中毒相容的时间尺度（寿命≈10 ms）。我们把这种由灾难性力量引发的脱轨率上升称为旅行债券。利用突变分析，我们阐明了这种亲和开关的机制。我们发现E9的n端区域与疏水性核心有稀疏接触，与E9的变构激活区（残基21-30）相连，其重塑引发免疫蛋白释放。通过引入适当位置的二硫化物桥来转移力传导途径，产生与集成技术测量的寿命相同的抗力复合物。E9中的跳闸开关对于其功能是理想的，因为它允许二部复合物亲和，因此宿主保护所需的稳定的粘菌素：免疫蛋白复合物可以很容易地转化为动力学不稳定的复合物，其解离是细胞入侵和竞争对手死亡所必需的。更一般地说，对E9:Im9复合物的两种力表型的观察表明，力可以重新塑造潜在的能量格局，为调节体内生物反应提供了新的机会；这使通常观察到的用动态力谱法和系综法测量的偏离率之间的差异变得合理。许多蛋白质与其他蛋白质相互作用是其功能的一部分。调节蛋白质复合物活性的一种方法是将它们分解。然而，一些复合物在动力学上是非常稳定的，目前还不清楚它们是如何在生物学相关的时间尺度上解离的。在这项研究中，我们利用大肠杆菌素E9（一种细菌毒素）与其免疫蛋白Im9之间的蛋白复合物来解决这个问题。这些高度活跃的复合体（寿命为几天）必须被分解，才能激活粘菌素。通过使用单分子力方法，我们发现，拉动colicin E9的一端会极大地破坏复合物的稳定，从而使其解离速度比其固有速率快100万倍。然后我们表明，防止这种不稳定（通过插入将E9的n端固定在适当位置的交联）产生动力学稳定的配合物。以前的假设是，力可以通过部分展开一个或多个结合伙伴来破坏蛋白质复合物的稳定性。我们的工作提供了新的实验证据，证明了这种情况，并为这种现象提供了一种机制，我们称之为旅行纽带。对于E9:Im9络合物，行程键行为允许稳定的络合物通过应用一个小得惊人的力迅速解离。
A single-molecule force study shows that rapid dissociation of a high-affinity protein interaction can be triggered by site-specific remodelling of one protein partner, and that prevention of remodelling maintains avidity. Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (Kd≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 106-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods. Many proteins interact with other proteins as part of their function. One method of modulating the activity of protein complexes is to break them apart. Some complexes, however, are extremely kinetically stable and it is unclear how these can dissociate on a biologically relevant timescale. In this study we address this question using protein complexes between colicin E9 (a bacterial toxin) and its immunity protein Im9. These highly avid complexes (with a lifetime of days) must be broken apart for colicin to be activated. By using single-molecule force methods we show that pulling on one end of colicin E9 drastically destabilises the complex so that it dissociates a million-fold faster than its intrinsic rate. We then show that preventing this destabilisation (by the insertion of cross-links that pin the N-terminus of E9 in place) yields a kinetically stable complex. It has previously been postulated that force can destabilise a protein complex by partially unfolding one or more binding partners. Our work provides new experimental evidence that shows this is the case and provides a mechanism for this phenomenon, which we term a trip bond. For the E9:Im9 complex, trip bond behaviour allows a stable complex to be rapidly dissociated by application of a surprisingly small force.
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DOI：
10.1073/pnas.070052697
发表时间：
2000-04-11
影响因子：
11.1
作者：
Baumgartner, W;Hinterdorfer, P;Drenckhahn, D
通讯作者：
Drenckhahn, D
影响因子：
5.6
作者：
Li, W;Keeble, AH;Kleanthous, C
通讯作者：
Kleanthous, C
影响因子：
64.8
作者：
CHOTHIA, C;JANIN, J
通讯作者：
JANIN, J
DOI：
10.1073/pnas.0404549101
发表时间：
2004-11-16
影响因子：
11.1
作者：
Dietz, H;Rief, M
通讯作者：
Rief, M
影响因子：
3.4
作者：
Evans, E;Ritchie, K
通讯作者：
Ritchie, K