Comparison of a structural and a functional epitope - PubMed (original) (raw)

. 1993 Dec 5;234(3):554-63.

doi: 10.1006/jmbi.1993.1611.

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Comparison of a structural and a functional epitope

B C Cunningham et al. J Mol Biol. 1993.

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Abstract

A comprehensive analysis of the energetic importance of the 31 side-chains buried at the interface between human growth hormone (hGH) and the extracellular binding domain of its receptor (hGHbp) has been carried out to assess the roles of contact side-chains in modulating the affinity and kinetics of binding. Each side-chain in hGH was converted to alanine, and the kinetics and affinity were measured using a biosensor device. This detects binding of the mutated hormones to the immobilized hGHbp by changes induced in refractive index. The data generated on the biosensor match affinities obtained by radio-immune assay in solution. The study shows that only one-quarter of the side-chains buried at the interface can account for the majority of the binding energy. These residues cluster near the center of the structural epitope. The role of these side-chains is predominantly to slow dissociation because most of the effect of the alanine substitutions is to increase the off-rate, not to slow the on-rate. The hormone associates about 10,000 times slower than expected from random diffusion but 1000 times faster than may be expected if one imposes strict orientation restraints for a productive collision. Electrostatic interactions partly modulate association because mutations at Arg residues most affect association and together contribute a factor of about 20 to the on-rate. The data suggest that the hormone and receptor associate by diffusion and electrostatics to form an ensemble of weak collisional complexes. From these a bound complex is produced that is stabilized by only a small proportion of the contacts. We suggest that solvation energies and/or side-chains interactions within the free hormone or receptor may be so favorable that little energy is gained at most side-chains upon binding. The fact that the functional binding epitope is much smaller than the structural epitope suggests it may be possible to design smaller hormone mimics.

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