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

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)] 2 β 2 type I enzyme is effectively inhibited by poly( L ‐proline), whereas the [α(II)] 2 β 2 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.