Related Experiment Videos
Ultrastructures and interactions of complement factors H and I
1Department of Immunology IMM 18, Scripps Research Institute, La Jolla, CA 92037.
Abstract:
The human complement regulatory protein, factor H, was examined by high resolution transmission electron microscopy. Results of electron microscopy confirm hydrodynamic analysis and indicate that factor H is a monomer of M(r) approximately 155,000. Factor H is an extended flexible molecule with a contour length of 495 A and a cross-sectional diameter of 34 A. Most images of factor H indicate that its polypeptide chain typically folds back on itself with the result that the average length of a factor H molecule is about half its contour length. Only one end of factor H associates with C3b. When bound to C3b, factor H still shows considerable conformational flexibility. Factor I is a bilobal protein of 130 A in length, and its two globular parts have maximal diameters of 54 and 49 A. The results establish that factor I is a two domain protein where the smaller subunit is a protease and the larger one is involved with binding C3b. Factor I binds C3b with a one-to-one stoichiometry in an ionic strength-dependent fashion. In the absence of sodium chloride an affinity constant of 5.7 x 10(5) M-1 was determined for factor I interaction with C3b. Whereas the Scatchard plot of factor I binding to C3b in the absence of factor H is linear, in the presence of factor H a curvilinear graph is obtained. The strong binding sites on C3b for factor I have an affinity at least 15-fold higher in the presence of factor H than in its absence. The results of both electron microscopy and binding studies were combined to compose a scheme envisioning how factors H and I cooperate for the processing of C3b.
Insights
High-resolution electron microscopy reveals the structure of complement factor H and its interaction with factor I and C3b. These findings elucidate the cooperative mechanism of complement regulation by factors H and I.
Area of Science:
- Complement system biology
- Structural biology
- Protein-protein interactions
Background:
- The human complement system is crucial for innate immunity but requires tight regulation to prevent self-damage.
- Factor H and Factor I are key regulatory proteins that control complement activation by cleaving C3b.
- Understanding the structure and interaction of these proteins is vital for comprehending complement-mediated diseases.
Purpose of the Study:
- To determine the molecular structure of human factor H using high-resolution transmission electron microscopy.
- To characterize the structural and binding properties of factor I.
- To elucidate the cooperative mechanism of action between factors H and I in the regulation of C3b.
Main Methods:
- High-resolution transmission electron microscopy (TEM) to visualize factor H and factor I.
- Hydrodynamic analysis to confirm molecular weight and shape.
- Binding studies to determine stoichiometry and affinity constants for factor I-C3b interactions.
- Scatchard analysis to assess binding in the presence and absence of factor H.
Main Results:
- Factor H is an extended, flexible monomer (approx. 155,000 M(r)) with a contour length of 495 Å, typically folding in half.
- Factor I is a bilobal protein (130 Å length) with distinct domains for C3b binding and protease activity.
- Factor H enhances the affinity of factor I for C3b by at least 15-fold, revealing a cooperative regulatory mechanism.
Conclusions:
- Factor H and Factor I cooperate to efficiently regulate the complement system.
- The structural insights into factor H and factor I provide a molecular basis for their regulatory functions.
- This study proposes a model for the combined action of factors H and I in C3b processing.