title: Assembly of the elongated collagen prolyl 4-hydroxylase α2β2heterotetramer around a central α2dimer.
abstract: Collagen prolyl 4-hydroxylase (C-P4H), an α2β2heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein-protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB-CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: M Kristian
authors.initials: MK
authors.lastname: Koski
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: Jothi
authors.initials: J
authors.lastname: Anantharajan
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.|Department of Biomedicine, University of Bergen, PO Box 7804, N-5009 Bergen, Norway.
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: Prathusha
authors.initials: P
authors.lastname: Dhavala
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: Abhinandan V
authors.initials: AV
authors.lastname: Murthy
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bergmann
authors.affiliation: Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, FIN-90014 Oulu, Finland rik.wierenga@oulu.fi.
authors.forename: Rik K
authors.initials: RK
authors.lastname: Wierenga
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D011392:Proline; C000591164:P4HA2 protein, human; D064828:Prolyl Hydroxylases
keywords: 2-oxoglutarate dioxygenase; collagen biosynthesis; extracellular matrix proteins; multidomain assembly; prolyl hydroxylase; small-angle X-ray scattering