Structural definition of the C5a C terminus by two-dimensional nuclear magnetic resonance spectroscopy

Abstract

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an alpha-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel alpha-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor.

Residues 21–30 within the Extracellular N-terminal Region of the C5a Receptor Represent a Binding Domain for the C5a Anaphylatoxin

“…On the other hand, the structured C5a produces well resolved two-dimensional NOESY spectra (7-9) with which perturbed resonances can be analyzed and assigned. In the presence of the receptor peptide, C5a and CGS-28805 exhibited a large number of the NOE connectivities characteristic of the three-dimensional structure of C5a (16,20). Except for shifts in the positions of a small number of cross-peaks, the NOESY spectra remain essentially unchanged (spectra not shown) without the intensity distortions or grossly broadened peaks seen with the spectrum of the receptor peptide (Fig.…”

“…Assignments of the proton resonances of C5a have been described previously (16,20). In short, the intraresidue spin systems were identified using through-bond connectivities observed in COSY or TOCSY type experiments.…”

“…Inappropriately accumulated C5a, on the other hand, stimulates smooth muscle contraction, causes vasodilation, increases vascular permeability, and can even recruit and stimulate granulocytes leading to the release of inflammatory molecules (2)(3)(4). The implication of C5a in various immune and inflammatory diseases prompted extensive structure-and-function studies (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In particular, NMR analysis defined C5a as being composed of a 4-helix core structure (residues 1-64) followed by a 10-residue C terminus with conformational variability (7)(8)(9)16).…”

“…The implication of C5a in various immune and inflammatory diseases prompted extensive structure-and-function studies (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In particular, NMR analysis defined C5a as being composed of a 4-helix core structure (residues 1-64) followed by a 10-residue C terminus with conformational variability (7)(8)(9)16). Determination of the three-dimensional structure of C5a followed by mutagenesis allowed the identification of many C5a residues required for receptor activation and others that may be more important for the structural integrity of the C5a protein (10 -15).…”

Random Mutagenesis of the Complement Factor 5a (C5a) Receptor N Terminus Provides a Structural Constraint for C5a Docking

“…The obtained structure was extended outward at the N terminus by manually adding a fragment corresponding to residues 24 -37 of C5aR in a position presumably allowing contacts with residue 27 of C5a. Using the Swiss PDB Viewer (www.expasy.org/spdbv/) (38), a structure of C5a taken from Protein Data Bank entry 1KJS (39) (22,40), the dihedral angles and of the peptide backbone for ligand residues 63-65 and 67 were manually adjusted within the allowable regions of the Ramachandran plot. The resulting docked model was subjected to energy minimization using the program SYBYL (Tripos, St. Louis, MO) to remove clashes between residues.…”