STRUCTURAL AND MUTATIONAL ANALYSIS OF AFFINITY-INERT CONTACT RESIDUESAT THE GROWTH HORMONE-RECEPTOR INTERFACE

Mutational studies have shown that over two-thirds of the contact side chains at the human growth hormone (hGH)-receptor interface have litt le or no impact on binding affinity when converted to alanine [Cunning ham, B. C., & Wells, J. A. (1993) J. Mol. Biol. 254, 554-563: Clackson , T., & Wells, J. A. (1995) Science 267, 383-386]. Herein, three of th e 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 dissociat ion rates are significantly affected and only slight, local disorder i s seen in the crystal structure. However, large and compensating chang es were observed in the enthalpy and entropy of binding as determined by isothermal titration calorimetry. The triple-alanine mutant bound w ith a more favorable enthalpy (Delta H = -12.2 +/- 0.7 kcal/mol) and c orresponding less favorable entropy [Delta S = -2.3 +/- 2.4 cal/(mol . K)] compared to the wild-type interaction [Delta H = -9.4 +/- 0.3 kca l/mol; Delta S = 7.7 +/- 1.2 cal/(mol . K)]. Dissection of the triple- alanine mutant into the single F25A and double Y42A/Q46A mutants showe d that the more favorable enthalpy was derived from the removal of the F25 side chain on helix-1 of the hormone. The Delta C-p values for bo th the triple-alanine mutant [-927 +/- 10 cal/(mol . K)] and the indiv idual mutants were significantly more negative than the Delta C-p for the wild-type interaction [-767 +/- 34 cal/(mol . K)]. Such negative D elta C-p values are consistent with the proposal that the hydrophobic effect is the primary contributor to the free energy of binding at thi s protein-protein interface. These results show that multiple-alanine mutations at contact residues may not affect binding kinetics, affinit y, or global structure, however, they can produce local structural cha nges and can cause large compensating effects on tile heal and entropy of binding. These studies emphasize that one cannot infer binding fre e energy from the existence of contacts alone and further support the notion that only a small set of contacts are critical for the human gr owth hormone-receptor interaction.