STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE 1 1 GROWTH-HORMONE/RECEPTORCOMPLEX REVEALS THE MOLECULAR-BASIS FOR RECEPTOR AFFINITY/

The designed G120R mutant of human growth hormone (hGH) is an antagoni st and can bind only one molecule of the growth hormone receptor. We h ave determined the crystal structure of the 1:1 complex between this m utant and the receptor extracellular domain (hGHbp) at 2.6 Angstrom re solution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall struct ure of the complex is very similar to the equivalent portion of the 1: 2 complex, showing that formation of the active complex does not invol ve major conformational changes. However, a segment involved in recept or-receptor interactions in the 1:2 complex is disordered in this stru cture, suggesting that its productive conformation is stabilized by re ceptor dimerization. The hormone binding site of the receptor comprise s a central hydrophobic patch dominated by Trp104 and Trp169, surround ed by a hydrophilic periphery containing several well-ordered water mo lecules. Previous alanine scanning showed that the hydrophobic ''hot s pot'' confers most of the binding energy. The new structural data, cou pled with binding and kinetic analysis of further mutants, indicate th at the hot spot is assembled cooperatively and that many residues cont ribute indirectly to binding. Several hydrophobic residues serve to or ient the key tryptophan residues; kinetic analysis suggests that Pro10 6 lacks the Trp104 main-chain into a required conformation. The electr ostatic contacts of Arg43 to hGH are less important than the intramole cular packing of its alkyl chain with Trp169. The true functional epit ope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacki ng interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periph ery can therefore attenuate the affinity of the central hot spot, perh aps reflecting a role in conferring specificity to the interaction. (C ) 1998 Academic Press Limited.