Engineering an interfacial zinc site to increase hormone-receptor affinity

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Human growth hormone (hGH) interacts with both hGH and human prolactin (hPRL) receptors.
• hGH binding to the hPRL receptor is tighter (50-fold) and zinc-dependent, unlike its binding to the hGH receptor.
• Prior research identified key residues in hGH and hPRL receptors involved in coordinating interfacial zinc.

Purpose of the Study:

• To investigate the possibility of engineering a metal-binding site into the hGH receptor.
• To enhance the binding affinity between hGH and its receptor.
• To explore the general principle of using interfacial metal-binding sites to improve protein-protein interactions.

Main Methods:

• Site-directed mutagenesis was used to introduce a putative zinc-coordinating residue (His) from the hPRL receptor into the hGH receptor at position 218 (Asn218-->His).
• The binding affinity of the engineered hGH receptor mutant for hGH was assessed in the presence of ZnCl2.
• Alanine-scanning mutagenesis was employed to characterize the hGH binding site on the mutant receptor.

Main Results:

• The Asn218-->His mutation in the hGH receptor resulted in a 20-fold increase in hGH binding affinity in the presence of Zn2+.
• The binding site on hGH for the engineered hGH receptor in the presence of zinc showed similarities to the hGH binding site for the hPRL receptor.
• This demonstrates successful transfer of metal-binding properties between homologous receptors.

Conclusions:

• The metal-binding site from the hPRL receptor can be functionally introduced into the homologous hGH receptor.
• Designing interfacial metal-binding sites represents a viable strategy for enhancing protein-protein binding affinity.
• These findings have implications for protein engineering and understanding receptor-ligand interactions.