Structure of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor: evidence for strong structural coupling between receptor binding sites

Abstract

The structure of the ternary complex between the phage display-optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 Å resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH v) contains 15 Site 1 mutations and binds >10 2 tighter to the hGHR ECD (hGH R1) at Site 1. It is biologically active and specific to hGHR. The hGH v Site 1 interface is somewhat smaller and 20 % more hydrophobic compared to the wild-type ( wt) counterpart. Of the ten hormone-receptor H-bonds in the site, only one is the same as in the wt complex. Additionally, several regions of hGH v structure move up to 9 Å in forming the interface. The contacts between the C-terminal domains of two receptor ECDs (hGH R1-hGH R2) are conserved; however, the large changes in Site 1 appear to cause global changes in the domains of hGH R1 that affect the hGH v-hGH R2 interface indirectly. This coupling is manifested by large changes in the conformation of groups participating in the Site 2 interaction and results in a structure for the site that is reorganized extensively. The hGH v-hGH R2 interface contains seven H-bonds, only one of which is found in the wt complex. Several groups on hGH v and hGH R2 undergo conformational changes of up to 8 Å. Asp116 of hGH v plays a central role in the reorganization of Site 2 by forming two new H-bonds to the side-chains of Trp104 R2 and Trp169 R2, which are the key binding determinants of the receptor. The fact that a different binding solution is possible for Site 2, where there were no mutations or binding selection pressures, indicates that the structural elements found in these molecules possess an inherent functional plasticity that enables them to bind to a wide variety of binding surfaces.