A Mutational Analysis of the Binding of Two Different Proteins to the Same Antibody
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- 作者：William Dall'Acqua ; Ellen R. Goldman ; Edward Eisenstein ; and Roy A. Mariuzza
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 30, 1996
- 年：1996
- 卷：35
- 期：30
- 页码：9667 - 9676
- 全文大小：574K
- 年卷期：v.35,no.30(July 30, 1996)
- ISSN：1520-4995
文摘
The crystal structures of the complexes between the anti-hen eggwhite lysozyme (HEL)antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2have shown that D1.3 contactsthese two proteins through essentially the same set of combining siteresidues [Fields, B. A., Goldbaum,F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature374, 739-742]. To probe the relativecontribution of individual residues to complex stabilization, singlealanine substitutions were introducedin the combining site of D1.3, and their effects on affinity for HELand for E5.2 were measured usingsurface plasmon resonance detection, fluorescence quench titration, orsedimentation equilibrium. Theenergetics of the binding to HEL are dominated by only 3 of the 13contact residues tested (
Gmutant-Gwild type > 2.5 kcal/mol):VLW92, VHD100, andVHY101. These form a patch at the center oftheinterface and are surrounded by residues whose apparent contributionsare much less pronounced (<1.5kcal/mol). This contrasts with the interaction of D1.3 with E5.2in which most the contact residues (11of 15) were found to play a significant role in ligand binding (>1.5kcal/mol). Furthermore, even thoughD1.3 contacts HEL and E5.2 in very similar ways, the functionallyimportant residues of D1.3 are differentfor the two interactions, with only substitutions at D1.3 positionsVH100 and VH101 greatlyaffectingbinding to both ligands. Thus, the same protein may recognizedifferent ligands in ways that are structurallysimilar yet energetically distinct.
Gmutant-Gwild type > 2.5 kcal/mol):VLW92, VHD100, andVHY101. These form a patch at the center oftheinterface and are surrounded by residues whose apparent contributionsare much less pronounced (<1.5kcal/mol). This contrasts with the interaction of D1.3 with E5.2in which most the contact residues (11of 15) were found to play a significant role in ligand binding (>1.5kcal/mol). Furthermore, even thoughD1.3 contacts HEL and E5.2 in very similar ways, the functionallyimportant residues of D1.3 are differentfor the two interactions, with only substitutions at D1.3 positionsVH100 and VH101 greatlyaffectingbinding to both ligands. Thus, the same protein may recognizedifferent ligands in ways that are structurallysimilar yet energetically distinct.