Structural investigation of protein:protein interactions within the membrane attack complex of complement

Our aim is to find out why some people are susceptible to bacterial infections, such as meningitis.
When our immune system detects the presence of infecting organisms, it triggers a rapid response to find, kill and remove the organisms. Complement is the name of one arm of our immune system and a remarkable feature of the complement response is its ability to kill organisms by piercing their cell membranes with many large holes, causing them to leak. These holes are made of five different proteins that rapidly self-assemble in an organised manner to form tube-like pores, called Membrane Attack Complexes (MAC), that pass through the cell membranes.
We are making the component parts of MAC proteins separately to determine their individual shapes and chemical characteristics, and to work out how they fit together. We aim to discover what features are important for the process of the MAC assembly and for maintaining a stable pore. Then we can determine how to stimulate, or to suppress, the assembly and stability of the pores.
This knowledge can be used to guide the design of drugs either to fight bacterial infections or to prevent unwanted complement attack, for example in auto-immune diseases, where a person’s immune system attacks their own body, or after transplant surgery to prevent organ rejection.

Our long-term strategic aim is to characterise module-module interactions involved in assembly and maintenance of the membrane attack complex (MAC), the lethal endpoint of an activated complement cascade. Stable formation of the MAC on target cells is required for protection against bacterial infections such as meningitis, however inappropriate deposition and activity of the MAC on host cells leads to several clinical pathologies involving tissue damage. There is little or no structural information available for the five proteins that comprise the MAC, nor are the interactions between them understood in any detail. We will attempt to address this dearth of structural data and thereby underpin research into therapeutic targeting of MAC assembly.
We will focus initially on delineating the interaction between the complement proteins C5 and C7, which is mediated by C-terminal domains from each protein - namely the C345C domain of C5 and the pair of factor I-like modules (FIMs) of C7. This interaction occurs in the fluid phase before C5 is activated to C5b, presumably as a strategy for accelerating the ensuing assembly process; recent work has shown that the interaction between these domains is also important for the subsequent steps in formation of a stable MAC. The complement control protein (CCP) modules, immediately N-terminal to the FIMs in the sequence of C7, have also been implicated in binding to activated C5b.
Building on our recent success in solving the solution structure of C5-C345C by NMR spectroscopy, we will initially determine the tertiary structure of the C7-FIMs ? thus providing the structure for a novel type of module. We will then use NMR as our primary tool, along with other biophysical techniques including isothermal titration calorimetry and FT-mass spectrometry, to characterise in depth the binding of C7-FIMs to C5-C345C. We aim to extract the kinetics and thermodynamics of binding, to map the interface of these two modules at atomic-level resolution and to ascertain any induced conformational changes. We will also determine the solution structure of the C7-CCPs by NMR spectroscopy and thus be in a position to reconstruct the structure of the functionally critical C-terminal half of C7. We will test our models for the binding mechanism by site-directed mutagenesis.