Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol.41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H(3) resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H(3) proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93L, in an ‘aromatic box’ of essentially tryptophan-93L, phenylalanine-34H and tyrosine-34L; asparagine-36L and tyrosine-34L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.
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Research Article|
August 01 1977
The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315
Steven K. Dower;
Steven K. Dower
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Simon Wain-Hobson;
Simon Wain-Hobson
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Peter Gettins;
Peter Gettins
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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David Givol;
David Givol
†Department of Chemical Immunology, Weizmann Institute for Science, Rehovot, Israel
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W. Roland C. Jackson;
W. Roland C. Jackson
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Stephen J. Perkins;
Stephen J. Perkins
‡Laboratory of Molecular Biophysics, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K.
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Christopher A. Sunderland;
Christopher A. Sunderland
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Brian J. Sutton;
Brian J. Sutton
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Carolyn E. Wright;
Carolyn E. Wright
‡Laboratory of Molecular Biophysics, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K.
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Raymond A. Dwek
Raymond A. Dwek
*Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1977 London: The Biochemical Society
1977
Biochem J (1977) 165 (2): 207–223.
Citation
Steven K. Dower, Simon Wain-Hobson, Peter Gettins, David Givol, W. Roland C. Jackson, Stephen J. Perkins, Christopher A. Sunderland, Brian J. Sutton, Carolyn E. Wright, Raymond A. Dwek; The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315. Biochem J 1 August 1977; 165 (2): 207–223. doi: https://doi.org/10.1042/bj1650207
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