title: Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.
abstract: Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase.
  
  
  BACKGROUND
  collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase
authors.affiliation: Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Myllyharju
authors.forename: K I
authors.initials: KI
authors.lastname: Kivirikko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D017354:Point Mutation; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D011392:Proline; D011393:Procollagen-Proline Dioxygenase