2 research outputs found
Assembly of the elongated collagen prolyl 4-hydroxylase Ī±2Ī²2 heterotetramer around a central Ī±2 dimer
Abstract
Collagen prolyl 4-hydroxylase (C-P4H), an Ī±2Ī²2 heterotetramer, is a crucial enzyme for collagen synthesis. The Ī±-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The Ī²-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 Ć
. The dimerization domains from the two Ī±-subunits form a proteināprotein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal Ī±-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/Ī²-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/Ī²-subunits locate at the far ends of the Ī²Ī±Ī±Ī² complex. The PDI/Ī²-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 Ī±-subunits has an important role in achieving the correct PSBāCAT 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
Structural enzymology binding studies of the peptideāsubstrateābinding domain of human collagen prolyl 4āhydroxylase (typeāII):high affinity peptides have a PxGP sequence motif
Abstract
The peptideāsubstrateābinding (PSB) domain of collagen prolyl 4āhydroxylase (CāP4H, an Ī±2Ī²2 tetramer) binds prolineārich procollagen peptides. This helical domain (the middle domain of the Ī± subunit) has an important role concerning the substrate binding properties of CāP4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the Cāterminal domain of the Ī± subunit). The crystal structures of the PSB domain of the human CāP4H isoform II (PSBāII) complexed with and without various short prolineārich peptides are described. The comparison with the previously determined PSBāI peptide complex structures shows that the CāP4HāI substrate peptide (PPG)3, has at most very weak affinity for PSBāII, although it binds with high affinity to PSBāI. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSBāII for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSBāI peptide complexes. In these PSBāII complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptideābinding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSBāI complex. These results suggest that PxGP (where x is not a proline) is the common motif of prolineārich peptide sequences that bind with high affinity to PSBāII