Torrey Pines Institute for Molecular Studies science image
Torrey Pines Institute for
Molecular Studies
3550 General Atomics Court, 2-129
San Diego, CA 92121-1122
USA
Scientists
Ronald Ogata
Member
Molecular Immunology

858.455.3736 - phone
858.455.3739 - fax

Protein-protein interactions in the complement system

Our work is focused on a group of over 30 proteins in the bloodstream that is collectively called complement. Binding of one or more of these proteins to specific molecular targets (cell surface molecules on foreign cells, for example) activates complement from its latent state and leads to an amplifying cascade of reactions involving multiple interactions among the complement proteins. These reactions generate chemically reactive proteins and protein complexes that mark foreign cells for ingestion by phagocytic cells and/or kill them directly. Activated complement proteins also potentiate the adaptive immune response and expedite clearance of immune complexes from the bloodstream.

Given these functions, it is not surprising that complement deficiency is associated with recurrent infections and autoimmune disease. However, complement is rather indiscriminate in its ability to attack cells., and uncontrolled or inappropriate activation can result in damage to host cells. This in turn can lead to harmful pathologies including acute and chronic inflammatory conditions such as ischemia reperfusion injury, autoimmune renal disease, rheumatoid arthritis, systemic lupus erythematosus, the vascular complications of diabetes, age-related macular degeneration, and Alzheimer's disease. Unfortunately, there are currently no drugs available that control complement activity and might therefore be used to restrict the effects of complement in these conditions.

Molecular interactions among the early constituents of the nascent MAC—C5b, C6, C7

One of the goals of our research program is to identify specific molecular interactions that take place during complement activation. Our current focus is on those interactions directly responsible for killing of foreign cells. This is carried out by a large, >1,000,000 kDa, complex of five complement proteins that kills by puncturing the plasma membrane of the invader. This complex, known as the complement membrane attack complex, or MAC, is composed of single copies of complement proteins C5b, C6, C7, and C8, and 10 or more copies of component C9. Each of these proteins is added individually, in numerical order, during MAC assembly. While the MAC composition and assembly process are known, the specific protein-protein interactions involved in assembling and maintaining the complex are largely unknown. Our current goal is to characterize at the amino acid level the specific sites of interaction between C5b and C6; between the resulting C5b,6 complex and C7; and between C5b,6,7 and C8. That is, we wish to identify and determine the spatial orientations of the amino acid residues in C5b and C6 that interact with each other within the C5b,6 complex, and to do the same with C7 and C8 in each succeeding complex.

Our ultimate goal is to obtain a high resolution 3D picture of each protein-protein interaction in C5b,6,7,8. This information will provide key leads in our future plans (see an example below) to design small molecule inhibitors of these early steps in MAC formation. As mentioned above, drugs that block MAC assembly are not currently available for clinical use but such drugs could provide healthcare benefits ranging from controlling inflammation to preventing the rejection of non-human transplanted organs (xenotransplantation).

These studies are difficult in part because of the large sizes and multiple binding activities of the MAC proteins themselves, and of their complexes. We have therefore adopted a strategy of producing structurally discrete segments or modules of the MAC proteins in bacteria. C6 and C7 in particular are very well suited to this strategy because each is composed entirely of a string of protein modules or domains. This strategy allows us to examine individual protein-protein interactions among C5b, C6, C7, and C8 in a simplified context, and provides enough material to determine binding activities by biochemical methods and even high resolution 3D structures.

We have already measured the binding functions of a 150 amino acid residue long domain from C5b (C345C) and two tandem 75 residue long FIM domains from C7; and in a collaborative study, Dr. Paul Barlow's laboratory at the University of Edinburgh recently solved the 3D structure of the C345C domain. We are now exploring the binding activities of other domains of C6 and C7, and using site-specific mutagenesis to identify the amino acid residues within interacting domains that mediate those interactions.

Peptidomimetic inhibitors of C5 activation

In earlier work, we identified a small, approximately 15 residue long segment of C5 (also present in C5b) that is essential for proteolytic activation of C5 to C5b. This segment appears to be a recognition site for the C5-activating protease (the C5 convertase), and indeed the corresponding synthetic peptide blocks C5 activation in vitro. The seg,emt lies within the C345C domain of C5 mentioned above, and the 3D structure determined by Dr. Barlow's laboratory indicates that the 15 residues form a hairpin that protrudes prominently from the b-barrel structure of the module.

To exploit this high resolution 3D picture of an apparent binding structure, we formed a collaboration with Dr. Adel Nefzi of TPIMS to design and construct small molecule mimics of this protrusion. Our hope is that such mimics will prevent C5 activation by competitive inhibition of the interaction between C5 and its convertase, and eventually lead to clinically useful small molecule inhibitors of C5 activation. Such inhibitors would offer the advantage of blocking many of the adverse effects of complement activation without compromising its beneficial functions.

Recent publications. 1-5

  1. Sandoval, A., R. Ai, J. M. Ostresh, and R. T. Ogata. 2000. Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5. J. Immunol. 165:1066-1073.
  2. Thai, C.-T., and R. T. Ogata. 2003. Expression and characterization of the C345C/NTR domains of complement components C3 and C5. J. Immunol. 171:6565-6573.
  3. Thai, C.-T., and R. T. Ogata. 2004. Complement Components C5 and C7: Recombinant Factor I Modules of C7 Bind to the C345C Domain of C5. J. Immunol. 173:4547-4552.
  4. Thai, C.-T., and R. T. Ogata. 2005. Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex. J. Immunol. in press.
  5. Bramham, J., C.-T. Thai, D. Soares, D. Uhrín, R. T. Ogata, and P. N. Barlow. 2005. Functional insights from the structure of the multifunctional C345C domain of C5 of complement. J. Biol. Chem. 280:10636-10645.

Publications

  1. Bramham, J., Thai, C.T., Soares, D., Uhrin, D., Ogata, R.T., Barlow, P.N. Functional insights from the structure of the multifunctional C345C domain of C5 of complement. J. Bio. Chem. 280:10636-10645, 2005
  2. Thai, C.T., Ogata, R. Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex. J. Immunol. 174:6227-6232, 2005.
  3. Bramham, J., Rance, M., Thai, C.T., Uhrin, D., Assa-Munt, N., Ogata, R.T., Barlow, P.N. 1H, 15N and 13C resonance assignments of the C345C domain of the complement component C5. J. Biomol. NMR 29:217-218, 2004.
  4. Thai, C.T., Ogata, R.T. Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5. J. Immunol. 173: 4547-4552, 2004.
  5. Nonaka, M.I., Hishikawa, Y., Moriyama, N., Koji, T., Ogata, R.T., Kudo, A., Kawakami, H., Nonaka, M. Complement C4b-binding protein as a novel murine epididymal secretory protein. Biol. Reprod. 69: 1931-1939, 2003.
  6. Thai, C.T., Ogata, R.T. Expression and characterization of the C345C/NTR domains of complement components C3 and C5. J. Immunol. 171:6565-6573, 2003.
  7. Richieri, G.V., Ogata, R.T., Zimmerman, A.W., Veerkamp, J.H., Kleinfeld, A.M. Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions. Biochemistry 39:7197-7204, 2000.
  8. Sandoval, A., Ai, R., Ostresh, J.M., Ogata, R.T. Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5. J. Immunol. 165:1066-1073, 2000.
  9. Low, P.J., Ai, R., Ogata, R.T. Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family. J. Immunol. 162:6580-6588, 1999. 
  10. Richieri, G.V., Low, P.J., Ogata, R.T., Kleinfeld, A.M. Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein. Biochemistry 38:5888-5895, 1999.
  11. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. Fatty acid interactions with native and mutant fatty acid binding proteins. Mol. Cell. Biochem. 192:77-85, 1999.
  12. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. The measurement of free fatty acid concentration with the fluorescent probe ADIFAB. Mol. Cell. Biochem. 192, 87-94, 1999.
  13. Ogata, R.T., Ai, R., Low, P.J. Active sites in complement component C3 mapped by mutations at indels. J. Immunol. 161:4785-4794, 1998.
  14. Richieri, G.V., Low, P.J., Ogata, R.T., Kleinfeld, A.M. Thermodynamics of fatty acid binding to engineered mutants of the adipocyte and intestinal fatty acid binding proteins. J. Biol. Chem. 273:7397-7405, 1998.
  15. Ogata, R.T., Low, P.J. Complement-inhibiting peptides identified by proximity to indels in the C3/4/5 protein family. J. Immunol. 158:3852-3860, 1997.
  16. Richieri, G.V., Low, P.J., Ogata, R.T., Kleinfeld, A.M. Mutants of rat intestinal fatty acid binding protein illustrate the critical role played by enthalpy-entropy compensation in ligand binding. J. Biol. Chem. 272:16737-16740, 1997.
  17. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. Kinetics of fatty acid interactions with fatty acid binding proteins from adipocyte, heart, and intestine. J. Biol. Chem. 271:11291-11300, 1996.
  18. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. Thermodynamics and kinetics of fatty acid interactions with rat liver fatty acid binding protein. J. Biol. Chem. 271:31068-31075, 1996.
  19. Ogata, R.T., Low, P.J. Complement component C5: Engineering of a mutant that is specifically cleaved by the C4-specific C1s protease. J. Immunol. 155:2642-2651, 1995.
  20. Ogata, R.T., Low, P.J., Kawakami, M. Substrate specificities of the protease of mouse serum Ra-reactive factor. J. Immunol. 154:2351-2357, 1995.
  21. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. Thermodynamics of fatty acid binding to fatty acid-binding proteins and fatty acid partition between water and membranes measured with the fluorescence probe ADIFAB. J. Biol. Chem. 270:15076-15084, 1995.
  22. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. Equilibrium constants for the binding of fatty acids with fatty acid binding proteins from adipocyte, intestine, heart, and liver; measured with the fluorescence probe ADIFAB. J. Biol. Chem. 269:23918-23930, 1994. 
  23. Richieri, G.V., Ogata, R.T., Kleinfeld, A.M. A fluorescently labeled intestinal fatty acid binding protein: Interactions with fatty acids and its use in monitoring free fatty acids. J. Biol. Chem. 267:23495-23501, 1992. 
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