The Functional Binding Epitope of a High Affinity Variant of Human Growth Hormone Mapped by Shotgun Alanine-scanning Mutagenesis: Insights into the Mechanisms Responsible for Improved Affinity

A high-affinity variant of human growth hormone (hGHv) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) ∼400-fold tighter than does wild-type (wt) hGH (hGHwt). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGHv binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGHv residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGHv differs significantly from that of hGHwt. The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding “hot spot”) remain important, but their contributions are attenuated in the hGHv interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGHwt are replaced by interactions that make positive contributions to the binding of hGHv. These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone–receptor interfaces. Thus, it appears that the improved binding affinity of hGHv site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.