Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase

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Abstract
Prolyl 4‐hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of ‐X‐Pro‐Gly‐ sequences and plays a central role in the synthesis of all collagens. The [α(I)]2β2 type I enzyme is effectively inhibited by poly(l‐proline), whereas the [α(II)]2β2 type II enzyme is not. We report here that the poly(l‐proline) and (Pro‐Pro‐Gly)10 peptide substrate‐binding domain of prolyl 4‐hydroxylase is distinct from the catalytic domain and consists of ∼100 amino acids. Peptides of 10–19 kDa beginning around residue 140 in the 517 residue α(I) subunit remained bound to poly(l‐proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the α(I) subunit residues 138–244 and expressed in Escherichia coli was soluble, became effectively bound to poly(l‐proline) agarose and could be eluted with (Pro‐Pro‐Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline‐rich peptide‐binding module. Studies with enzyme tetramers containing mutated α subunits demonstrated that the presence of a glutamate and a glutamine in the α(II) subunit in the positions corresponding to Ile182 and Tyr233 in the α(I) subunit explains most of the lack of poly(l‐proline) binding of the type II prolyl 4‐hydroxylase.