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• W4306682597 abstract "Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b’, and a’ domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a’ domain and tight hydrophobic interactions with the b’ domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer. Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact β/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b’, and a’ domains of the β/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a β-strand, which is the edge strand of its major antiparallel β-sheet. This extra region of the CAT domain interacts tightly with the β/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a’ domain and tight hydrophobic interactions with the b’ domain. Using this new information, the structure of the mature C-P4H-II α2β2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer. Collagen prolyl 4-hydroxylases (C-P4Hs; Enzyme Commission no.: 1.14.11.2), localized in the lumen of the endoplasmic reticulum, catalyze a vital cotranslational and post-translational modification of procollagen polypeptides required for the assembly of the collagen triple helix (1Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis.Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (326) Google Scholar, 2Myllyharju J. Prolyl 4–hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets.Ann. Med. 2008; 40: 402-417Crossref PubMed Scopus (173) Google Scholar, 3Gorres K.L. Raines R.T. Prolyl 4-hydroxylase.Crit. Rev. Biochem. Mol. Biol. 2010; 45: 106-124Crossref PubMed Scopus (399) Google Scholar, 4Salo A.M. Myllyharju J. Prolyl and lysyl hydroxylases in collagen synthesis.Exp. Dermatol. 2021; 30: 38-49Crossref PubMed Scopus (13) Google Scholar). C-P4Hs are Fe(II) and 2-oxoglutarate-dependent dioxygenases, which use molecular oxygen to hydroxylate proline residues that are present at the Y position of the -X-Y-Gly- repeats of the procollagen chain. The Fe(II) ion is bound in the active site to a fully conserved His-X-Asp--His sequence motif, being important for activating the O2 molecule. The catalysis involves oxidative decarboxylation of 2-oxoglutarate to succinate and carbon dioxide and the hydroxylation of procollagen peptidyl prolines into 4-hydroxyprolines. This modification is needed to provide stability for the triple helical collagen molecules. Subsequently, the collagens are secreted into the extracellular matrix where they are further modified and assembled into various supramolecular structures, and where they are involved in, for example, cell adhesion, cell migration, and cell remodeling during growth, differentiation, and wound healing (2Myllyharju J. Prolyl 4–hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets.Ann. Med. 2008; 40: 402-417Crossref PubMed Scopus (173) Google Scholar, 4Salo A.M. Myllyharju J. Prolyl and lysyl hydroxylases in collagen synthesis.Exp. Dermatol. 2021; 30: 38-49Crossref PubMed Scopus (13) Google Scholar, 5Shoulders M.D. Raines R.T. Collagen structure and stability.Annu. Rev. Biochem. 2009; 78: 929-958Crossref PubMed Scopus (2105) Google Scholar, 6Rappu P. Salo A.M. Myllyharju J.M. Heino J. Role of prolyl hydroxylation in the molecular interactions of collagens.Essays Biochem. 2019; 63: 325-335Crossref PubMed Scopus (41) Google Scholar). Collagens play also important roles in many pathological states, like fibrosis and cancer (2Myllyharju J. Prolyl 4–hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets.Ann. Med. 2008; 40: 402-417Crossref PubMed Scopus (173) Google Scholar, 7Franklin T.J. Therapeutic approaches to organ fibrosis.Int. J. Biochem. Cell Biol. 1997; 29: 79-89Crossref PubMed Scopus (109) Google Scholar, 8Vasta J.D. Raines R.T. Collagen Prolyl 4-hydroxylase as a therapeutic target.J. Med. Chem. 2018; 61: 10403-10411Crossref PubMed Scopus (35) Google Scholar, 9Shi R. Gao S. Zhang J. Xu J. Graham L.M. Yang X. et al.Collagen prolyl 4-hydroxylases modify tumor progression.Acta Biochim. Biophys. Sin. (Shanghai). 2021; 53: 805-814Crossref PubMed Scopus (10) Google Scholar). C-P4Hs are α2β2 heterotetramers (1Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis.Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (326) Google Scholar, 2Myllyharju J. Prolyl 4–hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets.Ann. Med. 2008; 40: 402-417Crossref PubMed Scopus (173) Google Scholar, 3Gorres K.L. Raines R.T. Prolyl 4-hydroxylase.Crit. Rev. Biochem. Mol. Biol. 2010; 45: 106-124Crossref PubMed Scopus (399) Google Scholar). The α-subunit contains the catalytic site for the C-P4H activity (10Myllyharju J. Kivirikko K.I. Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (164) Google Scholar), and the β-subunit is identical to protein disulfide isomerase (PDI, Enzyme Commission no.: 5.3.4.1), which is an enzyme and chaperone that functions in protein folding in the endoplasmic reticulum (11Elgaard L. Ruddock L.W. The human disulphide isomerase family: substrate interactions and functional properties.EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (625) Google Scholar, 12Hatahet F. Ruddock L.W. Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation.Antioxid. Redox Signal. 2009; 11: 2807-2850Crossref PubMed Scopus (502) Google Scholar, 13Khan H.A. Mutus B. Protein disulfide isomerase a multifunctional protein with multiple physiological roles.Front. Chem. 2014; 2: 70PubMed Google Scholar). In mammals, there are three isoforms of the α-subunit (Fig. 1) (14Helaakoski T. Annunen P. Vuori K. MacNeil I.A. Pihlajaniemi T. Kivirikko K.I. Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase α-subunit isoform: formation of an α2β2 tetramer with the protein disulfide-isomerase/β subunit.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4427-4431Crossref PubMed Google Scholar, 15Kukkola L. Hieta R. Kivirikko K.I. Myllyharju J. Identification and characterization of a third human, rat, and mouse collagen prolyl 4-hydroxylase isoenzyme.J. Biol. Chem. 2003; 278: 47685-47693Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), which are complexed with the same β-subunit, giving rise to three C-P4H tetramers, referred to as C-P4H-I, C-P4H-II, and C-P4H-III according to their α-subunit. Two splice variants have been characterized for both α(I) and α(II) (16Helaakoski T. Veijola J. Vuori K. Rehn M. Chow L.T. Taillon-Miller P. et al.Structure and expression of the human gene for the α subunit of prolyl 4-hydroxylase. The two alternatively spliced types of mRNA correspond to two homologous exons the sequences of which are expressed in a variety of tissues.J. Biol. Chem. 1994; 269: 27847-27854Abstract Full Text PDF PubMed Google Scholar, 17Nokelainen M. Nissi R. Kukkola L. Helaakoski T. Myllyharju J. Characterization of the human and mouse genes for the alpha subunit of type II prolyl 4-hydroxylase. Identification of a previously unknown alternatively spliced exon and its expression in various tissues.Eur. J. Biochem. 2001; 268: 5300-5309Crossref PubMed Scopus (19) Google Scholar) (Fig. S1). These splice variants have small sequence differences in the catalytic domain. C-P4H-I is the major isoenzyme, and its inactivation in mouse leads to early death during embryonic development, whereas mice lacking C-P4H-II are almost normal, but in combination with reduced amounts of C-P4H-I, show specific phenotypic abnormalities (18Holster T. Pakkanen O. Soininen R. Sormunen R. Nokelainen M. Kivirikko K.I. et al.Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase-I null mice.J. Biol. Chem. 2007; 282: 2512-2519Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 19Tolonen J.-P. Salo A.M. Finnilä M. Aro E. Karjalainen E. Ronkainen V.-P. et al.Reduced bone mass in collagen prolyl 4-hydroxylase P4ha1+/-;P4ha2-/- compound mutant mice.JBMR Plus. 2022; 6e10630Crossref PubMed Scopus (1) Google Scholar). C-P4H-I and C-P4H-II have different affinities for procollagen-like substrate peptides (20Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform α(II) and characterization of the type II enzyme tetramer. The α(I) andα(II) subunits do not form a mixed α(I)α(II)β2 tetramer.J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Myllyharju J. Kivirikko K.I. Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.EMBO J. 1999; 18: 306-312Crossref PubMed Scopus (61) Google Scholar). C-P4H-III is far less characterized than the first two isoforms (15Kukkola L. Hieta R. Kivirikko K.I. Myllyharju J. Identification and characterization of a third human, rat, and mouse collagen prolyl 4-hydroxylase isoenzyme.J. Biol. Chem. 2003; 278: 47685-47693Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and its in vivo role is as yet unknown. The α-subunits of human C-P4H-I and C-P4H-II share a sequence identity of about 65%, whereas α(I) and α(II) share about 35% sequence identity with α(III). The C-P4H α-subunit of each of the isoforms consists of four parts: the N-terminal (dimerization) domain, the peptide-substrate-binding (PSB) domain (middle domain), a linker region (L, of unknown structure and function), and the C-terminal catalytic domain (Fig. 2) (21Myllyharju J. Kivirikko K.I. Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.EMBO J. 1999; 18: 306-312Crossref PubMed Scopus (61) Google Scholar, 22Anantharajan J. Koski M.K. Kursula P. Hieta R. Bergmann U. Myllyharju J. et al.The structural motifs for the substrate binding and dimerization of the α subunit of collagen prolyl 4-hydroxylase.Structure. 2013; 21: 2107-2118Abstract Full Text Full Text PDF PubMed Google Scholar, 23Koski M.K. Anantharajan J. Kursula P. Dhavala P. Murthy A.V. Bergmann U. et al.Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.Biochem. J. 2017; 474: 751-769Crossref PubMed Scopus (12) Google Scholar). Crystal structures of the PSB domains of human C-P4H-I and C-P4H-II (24Pekkala M. Hieta R. Bergmann U. Kivirikko K.I. Wierenga R.K. Myllyharju J. The peptide-substrate-binding domain of collagen prolyl 4-hydroxylases is a tetratricopeptide repeat domain with functional aromatic residues.J. Biol. Chem. 2004; 279: 52255-52261Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 25Murthy A.V. Sulu R. Koski M.K. Tu H. Anantharajan J. Sah-Teli S.K. et al.Structural enzymology binding studies of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase (type-II): high affinity peptides have a PxGP sequence motif.Protein Sci. 2018; 27: 1692-1703Crossref PubMed Scopus (4) Google Scholar), with and without bound proline-rich peptides, show the mode of binding of these peptides in a groove lined by highly conserved tyrosine residues. Furthermore, a crystal structure of a construct consisting of the N-terminal (dimerization) domain and the PSB domain, which is referred as the double-domain construct (Fig. 2), shows how the N-terminal domain forms a protein–protein dimer interface via a coiled-coil helical dimerization motif, whereas the PSB domains point away from this interface (22Anantharajan J. Koski M.K. Kursula P. Hieta R. Bergmann U. Myllyharju J. et al.The structural motifs for the substrate binding and dimerization of the α subunit of collagen prolyl 4-hydroxylase.Structure. 2013; 21: 2107-2118Abstract Full Text Full Text PDF PubMed Google Scholar). The structure of the human C-P4H CAT domain is as yet unknown, but crystal structures of a monomeric Chlamydomonas reinhardtii P4H isoform 1 (Cr-P4H) (about 35% sequence identity with the human C-P4H-I CAT domain (Fig. 1)), both in its unliganded form (without peptide) and complexed with a peptide substrate, (Ser-Pro)5, have been solved (26Koski M.K. Hieta R. Böllner C. Kivirikko K.I. Myllyharju J. Wierenga R.K. The active site of an algal prolyl 4-hydroxylase has a large structural plasticity.J. Biol. Chem. 2007; 282: 37112-37123Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 27Koski M.K. Hieta R. Hirsilä M. Rönkä A. Myllyharju J. Wierenga R.K. The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif.J. Biol. Chem. 2009; 284: 25290-25301Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Like in all 2-oxoglutarate-dependent dioxygenases, the core of the fold consists of eight antiparallel β-strands (labeled as βI to βVIII), having the spiral topology of the double-stranded β-helix fold (the DSBH-fold) (28Dunwell J.M. Purvis A. Khuri S. Cupins: the most functionally diverse protein superfamily?.Phytochem. 2004; 65: 7-17Crossref PubMed Scopus (398) Google Scholar, 29Islam Md.S. Leissing T.M. Chowdhury R. Hopkins R.J. Schofield C.J. 2-oxoglutarate-dependent oxygenases.Annu. Rev. Biochem. 2018; 20: 659-672Google Scholar) and forming a major and a minor β-sheet. In the 2-oxoglutarate-dependent dioxygenases, the cofactor 2-oxoglutarate is bound in a deeply buried cavity between these two sheets, shielded from bulk solvent by the bound substrate molecule (28Dunwell J.M. Purvis A. Khuri S. Cupins: the most functionally diverse protein superfamily?.Phytochem. 2004; 65: 7-17Crossref PubMed Scopus (398) Google Scholar, 29Islam Md.S. Leissing T.M. Chowdhury R. Hopkins R.J. Schofield C.J. 2-oxoglutarate-dependent oxygenases.Annu. Rev. Biochem. 2018; 20: 659-672Google Scholar). In the structure of Cr-P4H complexed with its proline-rich substrate peptide, the peptide is bound in a tunnel shaped by two substrate-binding loops, being the hairpin loop and the βII-βIII loop (27Koski M.K. Hieta R. Hirsilä M. Rönkä A. Myllyharju J. Wierenga R.K. The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif.J. Biol. Chem. 2009; 284: 25290-25301Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the unliganded Cr-P4H structure, the substrate-binding loops are disordered and/or adopt different conformations (26Koski M.K. Hieta R. Böllner C. Kivirikko K.I. Myllyharju J. Wierenga R.K. The active site of an algal prolyl 4-hydroxylase has a large structural plasticity.J. Biol. Chem. 2007; 282: 37112-37123Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In addition, in the unliganded structure, the βI-βII catalytic loop (residues Tyr134 to Tyr140) adopts a different conformation when comparing liganded and unliganded structures. The conformational flexibility properties of the two substrate-binding loops and the catalytic loop are important for the catalytic function of this enzyme (27Koski M.K. Hieta R. Hirsilä M. Rönkä A. Myllyharju J. Wierenga R.K. The crystal structure of an algal prolyl 4-hydroxylase complexed with a proline-rich peptide reveals a novel buried tripeptide binding motif.J. Biol. Chem. 2009; 284: 25290-25301Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). PDI has a four-domain structure, referred to as the a, b, b’, and a’ domains (Fig. 2) (11Elgaard L. Ruddock L.W. The human disulphide isomerase family: substrate interactions and functional properties.EMBO Rep. 2005; 6: 28-32Crossref PubMed Scopus (625) Google Scholar). These domains have a thioredoxin fold. The a domain and a’ domain of PDI are also referred to as its catalytic domains, as they both have the conserved CGHC-sequence motif (Figs. 2 and S2). The cysteines of this motif are the CAT residues, needed to catalyze the formation, breaking, and isomerization of disulfide bridges of the PDI substrate proteins in their folding pathway. Studies have shown that the β/PDI subunit is required for providing the soluble and catalytically competent conformation of the α-subunit, but it is not understood how it is assembled with the α-subunit, how it is involved in intersubunit disulfide bridges, and what its role is in the procollagen hydroxylation reaction mechanism of the mature C-P4H α2β2 tetramer (30John D.C. Grant M.E. Bulleid N.J. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (95) Google Scholar, 31Vuori K. Pihlajaniemi T. Myllylä R. Kivirikko K.I. Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (128) Google Scholar, 32Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helaakoski T. Notbohm H. et al.The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide.EMBO J. 1999; 18: 65-74Crossref PubMed Scopus (54) Google Scholar, 33Pirneskoski A. Ruddock L.W. Klappa P. Freedman R.B. Kivirikko K.I. Koivunen P.J. Domains b' and a' of protein disulfide isomerase fulfill the minimum requirement for function as a subunit of prolyl 4-hydroxylase. The N-terminal domains a and b enhances this function and can be substituted in part by those of ERp57.Biol. Chem. 2001; 276: 11287-11293Abstract Full Text Full Text PDF Scopus (47) Google Scholar, 34Koivunen P. Salo K.E. Myllyharju J. Ruddock L.W. Three binding sites in protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase tetramer assembly.J. Biol. Chem. 2005; 280: 5227-5235Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The crystal structure of human PDI is known in oxidized (having a disulfide bond between the two cysteines of the CGHC-motif) and reduced (the cysteines of the CGHC motif are reduced) forms (35Wang C. Li W. Ren J. Fang J. Ke H. Gong W. et al.Structural insights into the redox-regulated dynamic conformations of human protein disulfide isomerase.Antioxid. Redox Signal. 2013; 19: 36-45Crossref PubMed Scopus (143) Google Scholar). Also known is the structure of the human heterodimer microsomal triglyceride transfer protein (MTP), which includes PDI as its β-subunit (36Biterova E.I. Isupov M.N. Keegan R.M. Lebedev A.A. Sohail A.A. Liaqat I. et al.The crystal structure of human microsomal triglyceride transfer protein.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 17251-17260Crossref PubMed Scopus (29) Google Scholar). The α-subunit of MTP is a lipid-binding protein, and its amino acid sequence and fold are not related to the α-subunit of C-P4H. In addition, the crystal structure of ERp57 (a PDI family member, present in the endoplasmic reticulum), complexed with tapasin (not related to the α-subunits of C-P4H and MTP), is known (37Dong G. Wearsch P.A. Peaper D.R. Cresswell P. Reinisch K.M. Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer.Immunity. 2009; 30: 21-32Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Small-angle X-ray scattering studies of mature C-P4H-I suggest that in the mature α2β2 C-P4H-I tetramer, the CAT domains of the two α-subunits point away from the α2-dimer interface (formed by the two N-terminal dimerization domains) and are capped by the β/PDI subunit, positioned at both ends of the elongated tetramer, suggesting that the β/PDI subunit interacts solely with the CAT domain (23Koski M.K. Anantharajan J. Kursula P. Dhavala P. Murthy A.V. Bergmann U. et al.Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.Biochem. J. 2017; 474: 751-769Crossref PubMed Scopus (12) Google Scholar). These small-angle X-ray scattering studies have provided also the shape information of proteolytically truncated forms of C-P4H-I, which were copurified together with the mature recombinant C-P4H-I. One of these truncated forms was a heterodimer complex of a truncated α-chain (consisting of the PSB and CAT domains but lacking the N-terminal dimerization domain) complexed with the intact β/PDI subunit. The truncation site of this heterodimer is near residue Asp139 of the α-subunit of C-P4H-I (just before the PSB domain) (Fig. 1) (23Koski M.K. Anantharajan J. Kursula P. Dhavala P. Murthy A.V. Bergmann U. et al.Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.Biochem. J. 2017; 474: 751-769Crossref PubMed Scopus (12) Google Scholar). The more detailed characterization of such a complex can provide key information on the structure of the CAT domain and its assembly with the β/PDI subunit, which is currently completely lacking. However, the optimal α-subunit truncation site for obtaining a heterodimer that has only the CAT domain of the α-subunit, complexed with the intact β/PDI subunit, is not clear, as it depends on the unknown structural and functional role of the linker region (Fig. 2). Here, we report on the characterization of two truncated human C-P4H-II variants in which the α-subunit is lacking either the N-terminal dimerization domain (C-P4H-II-Δ140) or the dimerization domain and the PSB domain (C-P4H-II-Δ281). These truncated variants of C-P4H-II are stable and soluble heterodimer complexes. The C-P4H-II-Δ281 complex could be crystallized, and its crystal structure reveals the structure of the CAT domain as well as its interactions with the β/PDI subunit. This crystal structure, together with the AlphaFold2 (DeepMind company and EMBL-EBI) (38Jumper J. Evans R. Pritzel A. Green T. Figurnov M. Ronnegerger O. et al.Highly accurate protein structure prediction with AlphaFold.Nature. 2021; 596: 583-589Crossref PubMed Scopus (4112) Google Scholar) model of the complete α-subunit dimer, enabled the prediction of the mode of assembly of the C-P4H-II α2β2 heterotetramer, which is also discussed. Considerable proteolytic degradation has been a problem in previous experiments on recombinant production of human C-P4H-I in the Escherichia coli Origami strain (23Koski M.K. Anantharajan J. Kursula P. Dhavala P. Murthy A.V. Bergmann U. et al.Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer.Biochem. J. 2017; 474: 751-769Crossref PubMed Scopus (12) Google Scholar). Therefore, the E. coli CyDisCo expression system (39Hatahet F. Nguyen V.D. Salo K.E. Ruddock L.W. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.Microb. Cell Fact. 2010; 9: 67Crossref PubMed Scopus (76) Google Scholar, 40Nguyen V.D. Hatahet F. Salo K.E. Enlund E. Zhang C. Ruddock L.W. Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli.Microb. Cell Fact. 2011; 10: 1Crossref PubMed Scopus (104) Google Scholar), which has been used for the expression of MTP for crystallization (36Biterova E.I. Isupov M.N. Keegan R.M. Lebedev A.A. Sohail A.A. Liaqat I. et al.The crystal structure of human microsomal triglyceride transfer protein.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 17251-17260Crossref PubMed Scopus (29) Google Scholar), was tested for production of human C-P4H-I and C-P4H-II. For this purpose, codon-optimized constructs of the α-subunit of C-P4H-I and C-P4H-II (Table S1) were coexpressed with β/PDI, being part of the CyDisCo expression vector. The complexes were expressed and purified as described in the Experimental procedures section. The C-P4H-I and C-P4H-II samples were pure after the size-exclusion chromatography (SEC) step. The yield of C-P4H-II was better, and proteolytic degradation was less than for C-P4H-I. Mass spectrometric (MS) peptide mapping of C-P4H-II confirmed that the purified C-P4H-II includes the N-terminal and C-terminal peptides of both subunits (Table S2). Therefore, subsequent experiments, aimed at finding the best possible truncation sites of the α-subunit that would allow for the recombinant generation and purification of a complex of the CAT domain assembled with β/PDI, were done with C-P4H-II. Four truncated constructs of the α-subunit were tested, referred to as C-P4H-II-Δ140, C-P4H-II-Δ281, C-P4H-II-Δ304, and C-P4H-II-Δ324, in which the first 140, 281, 304, and 324 residues, respectively, were deleted (Fig. 2). The truncated α-subunits were expressed with a 6× His tag at the N terminus, whereas the coexpressed β/PDI subunit was without a tag, identical as used for the expression of the mature C-P4H-I and C-P4H-II complexes (Table S1). Coexpression of PDI with the C-P4H-II-Δ140 construct (encoding for PSB and CAT, Fig. 2) as well as with the C-P4H-II-Δ281 construct (encoding for CAT only, Fig. 2) resulted in soluble complexes, whereas the C-P4H-II-Δ304 and C-P4H-II-Δ324 constructs were only expressed as insoluble proteins, which could not be purified. Apparently, the residues between 281 and 304 include amino acids that are required to form a soluble complex of the CAT domain, assembled with β/PDI. The C-P4H-II-Δ140 and C-P4H-II-Δ281 complexes were purified using the same protocols as used for mature C-P4H-I and C-P4H-II. The SDS-PAGE gel analysis showed that these samples are highly pure, and truncated α and intact β-subunits are present, which was also confirmed by MS characterization. Characterization by multiangle light scattering (MALS) (Fig. S3 and Table 1) confirmed that the purified C-P4H-II-Δ140 and C-P4H-II-Δ281 complexes are heterodimers. The CD spectra (Fig. S4A) show that wildtype C-P4H-II and its two truncated variants have the expected secondary structure properties. The CD melting curves of the truncated complexes (Fig. S4B) show Tm values of 50.2 and 53.2 °C for, respectively, C-P4H-II-Δ140 and C-P4H-II-Δ281, slightly higher than the Tm of mature C-P4H-II (Tm = 46.5 °C) (Table 1), which confirms that the truncated complexes indeed are stable proteins. Activity assays show that the two truncated variants have C-P4H activity, although to a lesser extent than mature C-P4H-II (Table 1). Subsequently, crystallization experiments were carried out with these purified complexes, which resulted in diffraction quality crystals of the C-P4H-Δ281 complex, as outlined in the next section.Table 1The molecular weights, the Tm values, and the activity assay results of mature C-P4H-II and its truncated variantsConstructMolecular weight (SEC–MALSaThe SEC–MALS graphs are shown in Fig. S3.)Tm (CDbThe CD melting curves are shown in Fig. S4B.)Indirect assaycThe mean values of the results of three experiments are provided. with 2-oxo[1-14C]glutarate and (PPG)10 substrate, measuring the hydroxylation coupled amount of formed14CO2Direct assaydThe results of two experiments are reported. The listed activity values obtained by the indirect and direct assays cannot be compared directly. with [14C]proline-labeled procollagen substrate, m" @default.
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• W4306682597 date "2022-12-01" @default.
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• W4306682597 title "Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer" @default.
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• W4306682597 doi "https://doi.org/10.1016/j.jbc.2022.102614" @default.
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