The Peptide-Substrate-binding Domain of Human Collagen Prolyl 4-Hydroxylases

Abstract

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers in which protein-disulfide isomerase serves as the ␤ subunit. Two isoforms of the catalytic ␣ subunit have been identified and shown to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue ␣(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H ␣(I) polypeptide The prolyl 4-hydroxylases (P4Hs) 1 catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in peptide linkages. Two P4H families are known today. The collagen P4Hs (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, have a central role in the synthesis of all collagens, the resulting 4-hydroxyproline residues being essential for assembly of the triple-helical molecules (1-3). The hypoxia-inducible factor (HIF) P4Hs, a family of cytoplasmic enzymes (4 -6), play a key role in the response of cells to hypoxia by catalyzing hydroxylation of the ␣ subunit of HIF. This subunit is synthesized continuously, and at least one of two critical proline residues becomes hydroxylated under normoxic conditions, the resulting 4-hydroxyproline being essential for rapid degradation of HIF-␣ (7-9). In hypoxia this hydroxylation ceases, HIF-␣ forms a dimer with HIF-␤, and the dimer then becomes bound to the HIF-responsive elements in a number of hypoxia-inducible genes.All vertebrate C-P4Hs are ␣ 2 ␤ 2 tetramers in which the ␤ subunit is identical to the enzyme and chaperone protein-disulfide isomerase (1-3). Two isoforms of the catalytic ␣ subunit have been characterized from human and mouse tissues and shown to form [␣(I)] 2 ␤ 2 and [␣(II)] 2 ␤ 2 tetramers, called the type I and type II C-P4Hs, respectively (10, 11). The type I enzyme is the most abundant form in most cells, but type II is the main form in chondrocytes, endothelial cells, and some other cell types (12, 13). The HIF-P4Hs appear to consist of only one type of monomer, the size of which ranges from 239 to 426 residues in the three human isoenzymes (4 -6).The C-P4Hs act on -Xaa-Pro-Gly-triplets in collagens and more than 15 other proteins with collagen-like sequences (1-3, 14), whereas the HIF-P4Hs hydroxylate -Leu-Xaa-Xaa-LeuAla-Pro-Tyr-and -Leu-Xaa-Xaa-Leu-Ala-Pro-Ala-sequences (7-9). All the P4Hs require Fe 2ϩ , 2-oxoglutarate, O 2 , and ascorbate, the 2-oxoglutarate being stoichiometrically decarboxylated during hydroxylation (1-3). The C-terminal regions of the C-P4H ␣ subunits and the HIF-P4H monomers contain four conserved residues, two histidines, and one aspartate that bind the Fe 2ϩ atom and a basic residue that binds the C-5 carboxyl group of the 2-oxoglutarate (15,16). This basic residue is a lysine ...

Catalytic Properties of the Asparaginyl Hydroxylase (FIH) in the Oxygen Sensing Pathway Are Distinct from Those of Its Prolyl 4-Hydroxylases

“…In the case of the HIF prolyl 4-hydroxylases, the minimum requirement is a peptide of more than 8 residues, whereas optimal interaction appears to require about 20 residues (23). The collagen prolyl 4-hydroxylases act even on tripeptides, although with very high K m values, long peptides being far better substrates (12)(13)(14)29). The present data and those reported previously (21) indicate that FIH requires particularly long peptide substrates (Table III).…”

The Peptide-Substrate-binding Domain of Collagen Prolyl 4-Hydroxylases Is a Tetratricopeptide Repeat Domain with Functional Aromatic Residues

“…The K d values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the ␣(I) and corresponding ␣(II) domains were very similar to the K m and K i values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The K d values determined for a 4-hydroxyproline-containing peptide indicated a marked decrease in the affinity of hydroxylated peptides for the domain (14). Many characteristic features of peptide binding to the type I and type II C-P4H tetramers can thus be explained by the binding to this domain rather than to the catalytic domain (14).…”

“…The K d values determined for a 4-hydroxyproline-containing peptide indicated a marked decrease in the affinity of hydroxylated peptides for the domain (14). Many characteristic features of peptide binding to the type I and type II C-P4H tetramers can thus be explained by the binding to this domain rather than to the catalytic domain (14).…”

“…NMR studies have shown that a recombinant human C-P4H ␣(I) polypeptide Phe 144 -Ser 244 forms a folded domain consisting of five ␣-helices and one short putative ␤-strand between the second and third ␣-helices (14); this structure is quite different from those of other proline-rich peptide binding modules, which consist mainly of ␤-strands (15)(16)(17)(18)(19)(20)(21)(22). Binding of the peptide (Pro-Pro-Gly) 2 to this domain caused major chemical shifts in many backbone amide resonances, with the residues showing the largest shifts being located mainly in helix ␣3 (14). The K d values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the ␣(I) and corresponding ␣(II) domains were very similar to the K m and K i values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers.…”

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

“…These PDI/ subunits are required to maintain the subunits in an active, soluble conformation and for retaining the tetramer within the ER. The subunit contains the catalytic site with conserved residues required for the binding of the Fe 2+ and 2-oxoglutarate (4,5) and the peptide-substrate-binding domain (6,7). Three subunit isoforms have been characterized from human and mouse that assemble into [ (I)] 2 2 , [ (II)] 2 2 and [ (III)] 2 2 tetramers (8-12) and display distinct tissue distributions (11,13,14).…”