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

Abstract

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are ␣ 2 ␤ 2 tetramers in which the catalytic ␣-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 Å resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five ␣-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich peptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-L-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90°a way from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a ␤-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the ␣ 2 ␤ 2 enzyme tetramer.

An Endoplasmic Reticulum Transmembrane Prolyl 4-Hydroxylase Is Induced by Hypoxia and Acts on Hypoxia-inducible Factor α

“…Many characteristic features of peptide binding to the C-P4Hs can be explained by binding to this domain rather than the catalytic domain (24). This domain has been crystallized and shown to belong to the tetratricopeptide repeat domains and have a groove lined by tyrosines, mutagenesis studies of which indicated that tyrosines 158, 163,193,196,199,230, and 233 are particularly important for peptide binding (25). This region of the P4H-TM sequence shows no similarity to those of the C-P4H ␣ subunits, and none of the seven tyrosines is conserved in the human P4H-TM sequence (Fig.…”

Structural and functional analysis of Nro1/Ett1: a protein involved in translation termination in S. cerevisiae and in O2-mediated gene control in S. pombe

“…1C; Pekkala et al 2004). The C-P4H TPR domain is involved in the binding of the [X-Pro-Gly] n triplets from collagen and enhances the activity of the C-P4H catalytic dioxygenase domain (also located within this a subunit) responsible for hydroxylation of the Pro side-chains from collagen.…”

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

“…TPR motifs mediate protein-protein and proteinpeptide interactions, and the assembly of multiprotein complexes (36). The structure of the peptide-substrate-binding domain of human collagen P4H-I, comprising residues 144-244 of the 517-amino-acid (I) subunit, has been recently shown to consist of two TPR motifs and a solvating helix (37). To study whether amino acid differences in the TPR motifs of the C. elegans and C. briggsae PHY polypeptides could explain the differences in their assembly properties, site directed mutagenesis was performed on two separate divergent amino acids in each of the PHY-1 and PHY-2 TPR motifs to change the C .…”