Comparison of a structural and a functional epitope.
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.
Cunningham
B C
BC
Department of Protein Engineering Genetech. Inc., South San Francisco, CA 94080-4990.
Wells
J A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000378:metabolism*
D000939:Epitopes; D011986:Receptors, Somatotropin; D013006:Growth Hormone