Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109.
PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer.
Instituto de Biomedicina de Valencia, C.S.I.C., Spain. jcalvete@ibv.csic.es
J J
JJ
Calvete
M A
MA
Campanero-Rhodes
M
M
Raida
L
L
Sanz
D000089:Acetic Anhydrides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002417:Cattle; D003512:Cyclohexanones / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D006493:Heparin / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D029584:Prostatic Secretory Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D012661:Semen / Q000737:chemistry*; D029589:Seminal Plasma Proteins; D016415:Sequence Alignment
D000089:Acetic Anhydrides; D003512:Cyclohexanones; D005353:Fibronectins; D029584:Prostatic Secretory Proteins; D011506:Proteins; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein; C031800:acetic anhydride; C018031:1,2-cyclohexanedione; D006493:Heparin; D001120:Arginine; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D008239:Lysine