Alanine Scanning Mutagenesis of the hGHR

buried at the core of the hGH-hGHR interface and makes van der Waals contact with several residues of hGH (K168, T175, and K172). A thorough alanine-scanning investigation of all of the Site 1 contact residues (30 side-chains) showed that a hydrophobic cluster involving W104 and W169 accounts for most of the free energy of binding (Fig. 5) (27). Importantly, it was also shown that the functionally significant residues on the hGHR are in direct contact with those on hGH.

The receptor also contains a centralized "functional epitope" surrounded by a number of hydrophilic residues that are generally less important for binding. Once again, it has been suggested that the nonfunctional, charged residues surrounding the very hydrophobic binding site are probably responsible for promoting solubility and specificity (27). In fact, it has been demonstrated that a single arginine residue (R43) in the extracellular domain of the hGHR contributes significantly to species specificity for growth hormone binding (28). Taken together, both of these mutational studies (on hGH and hGHR) clearly demonstrate that only a small subset of contact residues are important for modulating the energetics of binding.

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