The protein disulfide isomerase (PDI) family of folding catalysts are constructed from combinations of redoxactive and redox-inactive domains, all of which are probably based on the thioredoxin fold. To understand the function of each domain in the variety of catalytic reactions that each family member can perform (to differing extents), the domain boundaries of each family member must be known. By using a technique based on sequence alignments and the known structure of the a and b domains of human PDI, we generated a large number of domain constructs for all six redox-active human PDIs: PDI, PDIp, ERp72, ERp57, P5, and PDIr. The ability to generate significant amounts of soluble protein in E. coli from most of these domain constructs strongly indicates that the domain boundaries are correct. The implications for these domain boundaries on the tertiary structure of the human PDIs are discussed.
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.
Collagen prolyl 4-hydroxylases catalyze the hydroxylation of -X-Pro-Gly- sequences and play an essential role in the synthesis of all collagens. They require Fe(2+), 2-oxoglutarate, molecular oxygen and ascorbate, and all vertebrate collagen prolyl 4-hydroxylases are alpha(2)beta(2) tetramers. The alpha-subunits contain separate catalytic and peptide substrate-binding domains. Here, the crystallization of the peptide substrate-binding domain consisting of residues 144-244 of the 517-residue human alpha(I) subunit is described. The crystals are well ordered and diffract to at least 3 A. The space group is P3(1) or P3(2) and the asymmetric unit most probably contains a dimer.
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