The tertiary structure of calcium-bound Phl 7, a polcalcin, from timothy grass

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Polcalcins are small proteins found in the anthers and pollen of flowering plants. They are potent allergens and are surmised to be regulatory proteins of a currently unknown function. Polcalcin sequences range from 77 to 85 amino acids in length. The primary structure indicates that there are two EF-hand motifs (metal ion binding structural domains) and an additional α-helix located at the C-terminus. The crystal structure of calcium-bound Phl p 7 has previously been reported to be a domain-swapped dimer. However, some physical data suggest that calcium-bound Phl p 7 exists as a monomer under physiological conditions. Due to this discrepancy, the decision was made to determine the solution structure of calcium-bound Phl p 7. The structure calculations were performed in CYANA using the distance restraints and the predicted dihedral angles. The solution structure employed 2,176 distance restraints and 134 torsion angle restraints. The solution structure reveals a monomeric, ellipsoidal structure with an exposed potential target binding site lined with hydrophobic residues. 15N relaxation studies reveal that the protein has a rotational correlation time of 5.39 ns, supporting a monomeric protein. Additionally, the average order parameter value of the amide backbone vectors was found to be 0.90, indicating that the backbone is fairly rigid. These experimental results are consistent with calcium-bound Phl p 7 existing as a monomer under physiological conditions.