Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface.
The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14-20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k(on)) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (DeltaCp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.
Kouadio
Jean-Louis K
JL
Department of Biochemistry and Molecular Biology the University of Chicago, Chicago, Illinois 60637, USA.
Horn
James R
JR
Pal
Gabor
G
Kossiakoff
Anthony A
AA
D000409:Alanine / Q000235:genetics; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology*; D019281:Dimerization; D019277:Entropy; D015723:Gene Library; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
D011986:Receptors, Somatotropin; D019382:Human Growth Hormone; D000409:Alanine