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PDBsum entry 1dqq
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Immune system
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PDB id
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1dqq
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Three-Dimensional structures of the free and antigen-Bound FAB from monoclonal antilysozyme antibody hyhel-63(,).
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Authors
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Y.Li,
H.Li,
S.J.Smith-Gill,
R.A.Mariuzza.
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Ref.
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Biochemistry, 2000,
39,
6296-6309.
[DOI no: ]
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PubMed id
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Abstract
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Antigen-antibody complexes provide useful models for studying the structure and
energetics of protein-protein interactions. We report the cloning, bacterial
expression, and crystallization of the antigen-binding fragment (Fab) of the
anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and
antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed
with HEL was determined to 2.0 A resolution, while the structure of the unbound
antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In
the complex, 19 HyHEL-63 residues from all six complementarity-determining
regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous
polypeptide segments of the antigen. The interface also includes 11 bound water
molecules, 3 of which are completely buried in the complex. Comparison of the
structures of free and bound Fab HyHEL-63 reveals that several of the ordered
water molecules in the free antibody-combining site are retained and that
additional waters are added upon complex formation. The interface waters serve
to increase shape and chemical complementarity by filling cavities between the
interacting surfaces and by contributing to the hydrogen bonding network linking
the antigen and antibody. Complementarity is further enhanced by small (<3 A)
movements in the polypeptide backbones of certain antibody CDR loops, by
rearrangements of side chains in the interface, and by a slight shift in the
relative orientation of the V(L) and V(H) domains. The combining site residues
of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with
those of the free Fab, suggesting a loss in conformational entropy upon binding.
To probe the relative contribution of individual antigen residues to complex
stabilization, single alanine substitutions were introduced in the epitope of
HEL recognized by HyHEL-63, and their effects on antibody affinity were measured
using surface plasmon resonance. In agreement with the crystal structure, HEL
residues at the center of the interface that are buried in the complex
contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type)
> 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible
residues at the periphery are much less pronounced (<1.5 kcal/mol). In the
latter case, the mutations may be partially compensated by local rearrangements
in solvent structure that help preserve shape complementarity and the interface
hydrogen bonding network.
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