Biophysical and structural analysis of protein-protein interactions: from encounter complexes to computational design and directed evolution

蛋白质是细胞中的主力，参与执行大多数功能，如果不是全部功能的话。蛋白质如何做到这一点的一个核心特征是与其他蛋白质相互作用，这些蛋白质-蛋白质复合物传递的信息在所有其他相互作用同时进行的情况下被细胞解释。本申请旨在使用降解DNA（DNA酶）的细菌毒素与其抑制蛋白（称为免疫蛋白）的模型蛋白质-蛋白质相互作用（PPI），以解决有关PPI的基本问题，这些问题尚未得到解答，并且适用于大多数PPI。其中一个问题涉及蛋白质相互形成复合物的机制。所有的蛋白质复合物都要经过所谓的“遭遇复合物”，这是一个短暂的物种，在到达最终复合物的途中。这些遭遇复合物的性质已经被讨论和推测了很多年，但从来没有描述过这样一个复合物的结构，因为它们的性质是短暂的物种，在平衡条件下没有一个状态被大量填充。任何关于这种复合物的信息都将是对我们积累的关于蛋白质如何相互形成复合物的知识的宝贵补充。我们偶然发现了DNA酶-免疫蛋白复合物中的一种缔合机制，其中一种蛋白质围绕另一种蛋白质旋转，这为我们提供了一个机会，可以“冻结”许多方向，只留下一个可以在结构上定义的方向。这将通过在两个蛋白质之间插入一个稳定的（共价）键来实现，该键的位置可以捕获这些短暂相遇复合物之一并阻止其重新定向。然后，我们将尝试使用X射线晶体学获得这种共价复合物的三维结构。我们的目标是对许多复合体进行这种研究，从而获得更广泛的关于这种相遇复合体的图片。我们将解决的另一个问题涉及这些DNA酶-免疫蛋白复合物如何识别它们已经形成正确的关联（即特异性）。这是PPI中的一个普遍问题，因为细胞通常包含许多版本的蛋白质，这些蛋白质彼此非常相似;考虑到可能的伙伴关系的丰富性，它们的结合伙伴如何区分正确与错误？我们将从新描述的复合物的角度来解决这个问题，这些复合物来自合作者的实验室，其中已经设计了新的方法来重建PPI的特异性。在一组复合物中，特异性是由计算机算法设计的（计算设计），而在另一组复合物中，特异性是由达尔文选择在试管中设计的（定向进化）。我们的目标是通过我的实验室多年来开发的一系列生物物理和结构方法来验证这些新型复合物的特性，以便我们能够了解这些新型复合物与天然复合物的不同之处。这反过来将帮助我们了解是什么控制了这些复合物的特异性，以及我们与随意定制PPI特异性的距离有多近。

Proteins are the workhorses in a cell that are involved in carrying out most if not all of its functions. A central feature of how proteins do this is to interact with other proteins, with these protein-protein complexes conveying information that is interpreted by the cell in the context of all the other interactions going on simultaneously. This application aims to use a model protein-protein interaction (PPI) of a bacterial toxin that degrades DNA (a DNase) with its inhibitor protein, known as an immunity protein, to address fundamental questions concerning PPIs that remain unanswered and which are applicable to most PPIs. One of the questions concerns the mechanism by which proteins form complexes with each other. All protein complexes go through what is termed an 'encounter complex', a transient species en route to the final complex. The properties of these encounter complexes have been discussed and speculated on for many years but there has never been a description of the structure for such a complex because by their nature they are transient species where no one state is heavily populated under equilibrium conditions. Any information on such a complex would be a valuable addition to our accumulating knowledge on how proteins form complexes with each other. We have stumbled on a mechanism of association in DNase-immunity protein complexes where one protein pivots and rotates against the other, which offers us an opportunity of 'freezing-out' the numerous orientations and leaving just one that can be structurally defined. This will be accomplished by inserting a stable (covalent) bond between the two proteins in a position that traps one of these transient encounter complexes and stops it reorienting. We will then try to obtain the three dimensional structure of this covalent complex using X-ray crystallography. The aim is to do this for a number of complexes and so gain a broader picture of what this encounter complex looks like. The other question we will address relates to how these DNase-immunity protein complexes recognise they have formed the correct association (i.e. specificity). This is a generic problem in PPIs since cells often contain many versions of proteins that are very similar to each other; how do their binding partners distinguish right from wrong given the abundance of possible partnerships? We will address this question from the perspective of newly described complexes, which originate from laboratories of collaborators, where new methods have been devised to reconstruct the specificity of the PPI. In one set of complexes, specificity was design by a computer algorithm (computational design), while in another specificity was engineered by Darwinian selection in a test-tube (directed evolution). Our aim is to characterise the properties of these novel complexes through a series of biophysical and structural methods that my lab have developed over a number of years so that we can understand how these novel complexes differ from their natural counterparts. This in turn will help us understand what governs specificity in these complexes and how close we are to tailoring PPI specificity at will.