The high- and low-affinity receptor binding sites of growth hormone are allosterically coupled.

Growth hormone regulates its biological properties via a sequential hormone-induced receptor homodimerization mechanism. Using a mutagenesis-scanning analysis of 81 single and 32 pairwise double mutations, we show that the hormone's two spatially distal receptor binding sites (Site1 and Site2) are allosterically coupled. These allosteric effects are focused among a relatively few residues centered around the interaction between Asp-116 of the hormone and Trp-169 of the receptor in Site2. A rearrangement of this interaction triggered by mutations in Site1 produces both a major conformation and energetic reorganization of Site2, surprisingly without a reduction in overall binding affinity. Additionally, the data suggest a change in the conformational dynamics of several groups in Site2 that appear to be important in defining the Site2 interaction. Changes in binding energy of the affected Site2 residues usually range in magnitude from 3- to 60-fold, but in one case are as large as 10(4).

[1]  D. Myszka,et al.  CLAMP: a biosensor kinetic data analysis program. , 1998, Trends in biochemical sciences.

[2]  W. Windsor,et al.  Crystal structure of a complex between interferon-γ and its soluble high-affinity receptor , 1995, Nature.

[3]  H. Heyneker,et al.  High-level secretion of human growth hormone by Escherichia coli. , 1987, Gene.

[4]  S. Sprang,et al.  Cytokine structural taxonomy and mechanisms of receptor engagement , 1993 .

[5]  Charles Eigenbrot,et al.  Crystal Structure at 1.7 Å Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor , 1997, Cell.

[6]  A. D. de Vos,et al.  Hematopoietic receptor complexes. , 1996, Annual review of biochemistry.

[7]  S. Atwell,et al.  Structural plasticity in a remodeled protein-protein interface. , 1997, Science.

[8]  S. Bass,et al.  The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. , 1990, The Journal of biological chemistry.

[9]  A. Kossiakoff,et al.  Site2 binding energetics of the regulatory step of growth hormone–induced receptor homodimerization , 2003, Protein science : a publication of the Protein Society.

[10]  A. Kossiakoff,et al.  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. , 2003, Journal of molecular biology.

[11]  H. Lowman,et al.  Affinity maturation of human growth hormone by monovalent phage display. , 1993, Journal of molecular biology.

[12]  David Baker,et al.  Convergent mechanisms for recognition of divergent cytokines by the shared signaling receptor gp130. , 2003, Molecular cell.

[13]  M. Ultsch,et al.  Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors , 1994, Protein science : a publication of the Protein Society.

[14]  M. Ultsch,et al.  Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. , 1992, Science.

[15]  J. Wells,et al.  Systematic mutational analyses of protein-protein interfaces. , 1991, Methods in enzymology.

[16]  David W. Banner,et al.  The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor , 1996, Nature.

[17]  M. Ultsch,et al.  The X-ray structure of a growth hormone–prolactin receptor complex , 1994, Nature.

[18]  M. Dwyer,et al.  Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries. , 2004, Biochemistry.

[19]  Anthony A Kossiakoff,et al.  Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Schiffer,et al.  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. , 2002, Journal of molecular biology.

[21]  Robert M. Stroud,et al.  Efficiency of signalling through cytokine receptors depends critically on receptor orientation , 1998, Nature.

[22]  T. Clackson,et al.  Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity. , 1998, Journal of molecular biology.

[23]  M. Doyle,et al.  Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface. , 1998, Analytical biochemistry.

[24]  A. D. de Vos,et al.  Structural basis for cytokine hormone-receptor recognition and receptor activation. , 1998, Advances in protein chemistry.

[25]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Bazan,et al.  Structural design and molecular evolution of a cytokine receptor superfamily. , 1990, Proceedings of the National Academy of Sciences of the United States of America.