Random mutagenesis of the complement factor 5a (C5a) receptor N terminus provides a structural constraint for C5a...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) are vital cell surface receptors involved in numerous physiological processes.
• The N-terminus of GPCRs often plays a critical role in ligand binding and receptor activation.
• The complement factor 5a receptor (C5aR) is a key mediator of inflammatory responses.

Purpose of the Study:

• To identify essential residues in the N terminus of the human C5aR involved in C5a ligand binding.
• To elucidate the structural basis of the C5a/C5aR interaction.
• To provide a foundation for the rational design of C5aR-targeting therapeutics.

Main Methods:

• Random saturation mutagenesis of the C5aR N terminus.
• Functional screening of mutant receptors for activation by C5a.
• Computational docking to model ligand-receptor interactions.
• Rescreening with a modified C5a ligand (C5a C27R) to assess disulfide bond involvement.

Main Results:

• Residues 24-30 of C5aR showed a propensity to mutate to cysteine, suggesting disulfide bond formation with C5a Cys27.
• Computational modeling supported a model of C5a/C5aR interaction involving disulfide bonds.
• When disulfide bond formation was prevented, no single N-terminal residue was essential, but region 19-29 remained conserved in functional mutants.
• This indicates that the 19-29 region of C5aR is critical for productive interaction with C5a.

Conclusions:

• The N terminus of C5aR, particularly residues 19-29, is essential for high-affinity binding and signaling of the C5a ligand.
• Disulfide interactions between C5a and C5aR may contribute to the initial binding event.
• Understanding these interactions can inform the development of selective modulators of C5aR activity.