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

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.

1.Structure of a protein-protein interaction (PPI) encounter complex. All protein-protein associations go through an encounter complex prior to forming the final complex but as yet there has been no atomic-level resolution of such an intermediate. Our finding that encounter complexes of colicin DNases with Im proteins are rotamers suggests a way towards achieving this difficult goal by freezing-out (non-cognate) complexes by disulfide bond engineering. Covalent complexes will be crystallized, their structures solved and analysed. 2. Dissection of the kinetics and thermodynamics of computationally redesigned protein-protein complexes. One of the major advances in this area has come from David Baker (US) whose lab have developed a novel second site suppressor strategy for engineering novel PPI specificities, using colicin DNase-Im protein complexes as the model system. These novel complexes, many of which remain to be characterised, have been provided to us and will be analysed in terms of binding affinity, specificity and energetics. 3. Structural and biophysical analysis of directed evolution-derived PPIs. Directed evolution is used for investigating the evolution of protein function and for generating new functions. The methods that are generally in use however tend only to select for improvements in binding. In vitro compartmentalisation is a relatively new method where other functions such as enzyme inhibition can also be selected. Using this method novel colicin DNase immunity protein variants have been generated by Dan Tawfik's lab (Israel) that have been provided to us. We will conduct biophysical and structural studies on these novel complexes that will provide the ultimate test of the current specificity models we have proposed for these and other divergently evolved protein complexes.