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

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.