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Biochemistry. 1996 Aug 13;35(32):10300-7.

Structural and mutational analysis of affinity-inert contact residues at the growth hormone-receptor interface.

Author information

1
Department of Protein Engineering. Genentech, Inc., South San Francisco, California 94080, USA.

Abstract

Mutational studies have shown that over two-thirds of the contact side chains at the human growth hormone (hGH)-receptor interface have little or no impact on binding affinity when converted to alanine [Cunningham, B. C., & Wells, J. A. (1993) J. Mol. Biol. 234, 554-563; Clackson, T., & Wells, J. A. (1995) Science 267. 383-386]. Herein, three of the most buried, yet functionally inert, residues on hGH (F25, Y42, and Q46) have been simultaneously mutated to alanine. Binding kinetics of the triple-alanine mutant shows that neither association nor dissociation rates are significantly affected and only slight, local disorder is seen in the crystal structure. However, large and compensating changes were observed in the enthalpy and entropy of binding as determined by isothermal titration calorimetry. The triple-alanine mutant bound with a more favorable enthalpy (delta H = -12.2 +/- 0.7 kcal/mol) and corresponding less favorable entropy [delta S = -2.3 +/- 2.4 cal/(mol.K)] compared to the wild-type interaction [delta H = -9.4 +/- 0.3 kcal/mol; delta S = 7.7 +/- 1.2 cal/(mol.K)]. Dissection of the triple-alanine mutant into the single F25A and double Y42A/Q46A mutant showed that the more favorable enthalpy was derived from the removal of the F25 side chain on helix-1 of the hormone. The delta Cp values for both the triple-alanine mutant [-927 +/- 10 cal/(mol.K)] and the individual mutants were significantly more negative than the delta Cp for the wild-type interaction [-767 +/- 34 cal/(mol.K)]. Such negative delta Cp values are consistent with the proposal that the hydrophobic effect is the primary contributor to the free energy of binding at this protein-protein interface. These results show that multiple-alanine mutations at contact residues may not affect binding kinetics, affinity, or global structure; however, they can produce local structural changes and can cause large compensating effects on the heat and entropy of binding. These studies emphasize that one cannot infer binding free energy from the existence of contacts alone and further support the notion that only a small set of contacts are crucial for the human growth hormone-receptor interaction.

PMID:
8756685
DOI:
10.1021/bi960513b
[Indexed for MEDLINE]

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