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Human complement protein C9 is a calcium binding protein. Structural and functional implications
N M Thielens1, K Lohner, A F Esser
1Department of Comparative and Experimental Pathology, University of Florida, Gainesville 32610.
Insights
Human complement protein C9 binds calcium, which is essential for its thermal stability. Removing calcium (Ca2+) leads to aggregation and loss of hemolytic activity, highlighting calcium's regulatory role.
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- Human complement protein C9 is a key component of the membrane attack complex.
- The role of metal ions in C9 function and stability is not fully understood.
Purpose of the Study:
- To investigate the binding of calcium (Ca2+) to human complement protein C9.
- To determine the functional and structural consequences of calcium binding and removal.
Main Methods:
- Equilibrium dialysis was used to measure Ca2+ binding affinity.
- Ethylenediaminetetraacetic acid (EDTA) was used to chelate Ca2+.
- Thermal stability and hemolytic activity assays were performed.
- Ligand blotting identified the Ca2+ binding site.
Main Results:
- Human C9 binds 1 mol of Ca2+ per mole of protein with a dissociation constant of 3 μM.
- Calcium removal by EDTA decreases C9's thermal stability, leading to aggregation and loss of hemolytic activity upon heating.
- Protein aggregation due to calcium removal can be prevented by stabilizing salts.
- The Ca2+ binding site is located in the amino-terminal half of C9.
- Calcium removal does not affect C9's hemolytic activity or binding to C8 or lipid vesicles at physiological temperatures.
Conclusions:
- Bound calcium provides essential thermal stability to human complement protein C9.
- Calcium may play a regulatory role in C9's membrane insertion function.
- Further research is needed to elucidate the precise structural and functional role of Ca2+ in C9.
Abstract:
Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.