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

Abstract

The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α β tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described. The comparison with the previously determined PSB-I peptide complex structures shows that the C-P4H-I substrate peptide (PPG) , has at most very weak affinity for PSB-II, although it binds with high affinity to PSB-I. The replacement of the middle PPG triplet of (PPG) to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB-II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB-I peptide complexes. In these PSB-II 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 peptide-binding 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 PSB-I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline-rich peptide sequences that bind with high affinity to PSB-II.

Prolyl and lysyl hydroxylases in collagen synthesis

“…[67] A high-resolution crystal structure is not yet available for the collagen P4H tetramer or for its catalytic α subunit due to the insolubility of the latter. However, a small-angle X-ray scattering (SAXS) model for the entire tetramer [68] as well as high-resolution crystal structures of two of the three functional domains of the α subunit, the N-terminal dimerization domain and the central peptide substrate binding domain, [68][69][70][71] and the PDI [72] have been solved. Based on the SAXS model the collagen P4H tetramer is an extended symmetric molecule with two lobes and a total length of 290 Å.…”

Role of prolyl hydroxylation in the molecular interactions of collagens

“…There is also a marked difference between the two enzymes in the inhibition by a competitive inhibitor poly(L-proline) [17,25]. A peptide-substrate-binding domain has been identified in the C-P4H α subunits and there are distinct differences in the binding properties and structure of this domain in the C-P4H-I and II [26,27].…”

Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer

“…2 ) ( 21 , 22 , 23 ). Crystal structures of the PSB domains of human C-P4H-I and C-P4H-II ( 24 , 25 ), 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.…”