Structural biology of soft tick complement inhibitor (OMCI) alone and in complex with C5

The complement system is a group of approx. 30 proteins that act together to form a frontline defence against invasion by parasites and other pathogens. As for all proteins we can only really understand the ways in which these proteins act to combat invasion by knowledge of their detailed, atomic, structure. However, many of the complement system proteins are difficult to study at this level of detail and we therefore have little atomic level structural information for the central components of this important biological system. Most parasites attempt to overcome the defence mounted by the complement system by expressing molecules that act specifically to interfere with these defences. This application seeks to use an anti-complement molecule expressed by a tick to increase our understanding both of how the tick protein works (by solving the atomic structure of this inhibitor) and also to increase our understanding of one of the key complement components, a protein called C5, by discovering precisely how the tick inhibitors prevent its correct functioning. A vital first step towards this aim is the identification of the site of interaction between C5 and the inhibitor. We will use a variety of different biochemical methods to identify the site of interaction. Including (1) determining which fragments of C5 bind to the inhibitor, (2) studying the interaction between C5 and inhibitor in competition with complement components which bind to C5, and (3) introducing single amino acid changes to C5 that prevent the inhibitor binding. Armed with these data and materials (purified fragments of C5) we will attempt to determine an atomic structure for a complex between C5 (or a significant part of it) and the tick inhibitor to give us a framework on which to interpret our biochemical data and prior knowledge of the biological function of C5.

Complement (C) is an integral part of the vertebrate immune system that forms a frontline defence against parasites and orchestrates the subsequent acquired immune response. C comprises about 30 fluid-phase and membrane-associated proteins and its activation results in the release of acute proinflammatory mediators and formation of the terminal membrane attack complex (MAC) which can lyse cells directly or trigger a variety of cellular responses. Activation of C occurs via a tightly regulated proteolytic cascade dependent on conformational changes to the three dimensional structure of proteins induced by multi-protein complex formation and the cleavage events themselves. Most parasites express specific inhibitory proteins, produce physical barriers and/or sequester host regulatory molecules to counteract the harmful effects of activated C. A pivotal event in the activation of C is cleavage of protein C5 releasing the proinflammatory peptide C5a and generating protein C5b which acts as the nucleus for the assembly of the MAC. We have recently expressed a small protein inhibitor from soft tick saliva that specifically binds C5 and prevents C5a release and MAC formation. The inhibitor is the first natural molecule known to target C5 and offers an excellent opportunity to understand more about the normal operation of C5, since the site or sites blocked by the inhibitor (which is roughly 10 times smaller than C5) are obviously critical to C5 function. Thus the aim of this proposal is to characterise the interaction between the inhibitor and C5 and obtain structural data enabling an atomic description of the interaction between the two proteins. The C5 region to which the inhibitor binds will be defined using a variety of biochemical techniques (mass spectrometry, surface plasmon resonance, radioblot) and by identifying peptides corresponding to specific regions of C5 that bind to the inhibitor and prevent its action. Mutation of specific amino acid residues in the C5 will be undertaken to provide additional evidence for the involvement of a given region in binding. If the C5-inhibitor complex is as tightly binding (KD in low nanomolar range), as we believe, our method of choice will be to purify the fully occupied complex before attempting crystallisation. If this proves unsuccessful, we will mix together OmCI and recombinant C5 regions that interact with OmCI at appropriate stoichiometry immediately before crystallisation. A robot will be used to screen a wide variety of conditions (up to 600) to obtain crystals of the inhibitor-C5 complex. Crystals will be exposed to an intense X-ray source and the resulting diffraction pattern used to derive the three dimensional coordinates of the atoms that form the inhibitor - C5 complex.