Fig. 5. Space filling model of the structure of the 1:1 complex of hGH and the hGHR. (Figure reproduced from ref. 27 with permission; see ref. 27 for color figure.) The two molecules are separated to show the energetic contribution to binding of individual residues. hGH is shown with residues at the Site 1 interface shaded in darker gray. One of the more important residues, K172, is labeled. The hGHR interface is shown with all Site 1 contact residues shaded in darker gray. W104, which is labeled, is one of the two most important residues on hGHR for binding hGH.

binding. Using this technique, dissociation and association rates (® 2 x 10-3 s1 and « 3 x 105 M-1s-1, respectively for wild-type hGH) were determined for alanine mutations at all residues in the receptor interface. This study showed that only about 25% of the residues on hGH, which are known from the crystal structure to be in contact with the hGHR, are actually important for binding (Fig. 5). These residues (predominantly L45, R64, K172, T175, F176, and R178) cluster at the center of the receptor contact interface or the "structural epitope;" collectively, these energetically important residues are referred to as the "functional epitope." Mostly, mutations at these residues cause a faster dissociation rate, which means that these residues function to stabilize the bound complex by slowing hGH dissociation.

Because many of the affinity-inert contact residues are polar or charged, it is possible that these side-chains are important for maintaining specificity and solubility. Interestingly, mutation of some of these affinity-inert residues has been shown to have large yet compensating effects on the enthalpy and entropy of binding (12).

Based on fluorescence-quenching experiments, it was originally speculated that a tryptophan residue in the extracellular domain of hGHR was involved in hGH binding (26). Additionally, mutation of W104 resulted in a very large decrease in binding affinity for hGH (about 2500-fold) (26). Further insight into the importance of W104 was gained with the crystal structure of the hGH-hGHR complex (1). In Site 1, this residue is highly

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