Related Experiment Videos
Structure and specificity of the anti-digoxin antibody 40-50
P D Jeffrey1, J F Schildbach, C Y Chang
1Department of Macromolecular Crystallography, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
Insights
The anti-digoxin antibody 40-50 Fab exhibits unique sequence and specificity, distinct from other high-affinity antibodies. Its three-dimensional structure reveals a binding pocket that explains its precise recognition of cardenolides.
Area of Science:
- Structural biology
- Immunology
- Pharmacology
Background:
- High-affinity antibodies against digoxin are crucial for treating digoxin toxicity.
- Understanding antibody-hapten interactions at a molecular level is key to developing targeted therapeutics.
Purpose of the Study:
- To determine the sequence, specificity, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain.
- To elucidate the molecular basis for the antibody's fine specificity towards cardenolides.
Main Methods:
- X-ray crystallography was used to determine the three-dimensional structure of the 40-50 Fab-ouabain complex.
- Binding constants were measured for various digoxin analogs to assess antibody specificity.
- Sequence homology analysis was performed comparing antibody 40-50 to other anti-digoxin antibodies.
Main Results:
- The 40-50 antibody shows no close sequence homology with other high-affinity anti-digoxin antibodies.
- A distinct specificity pattern was observed for structurally different digoxin analogs.
- The crystal structure revealed that the hapten (ouabain) is bound in a groove-like pocket, involving five of six complementarity-determining regions.
- The antibody-hapten interface is characterized by significant surface complementarity and specific hydrogen bonds, enabling selective binding.
Conclusions:
- The immune system can generate antibodies with diverse structural solutions for binding small molecules like cardenolides.
- The determined structure of the 40-50 Fab-ouabain complex provides a molecular explanation for the antibody's observed fine specificities.
- Comparison with other anti-digoxin antibody structures highlights different binding orientations for related haptens, underscoring antibody diversity.
Abstract:
We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.