The Crystal Structure of an Algal Prolyl 4-Hydroxylase Complexed with a Proline-rich Peptide Reveals a Novel Buried Tripeptide Binding Motif

Abstract

Plant and algal prolyl 4-hydroxylases (P4Hs) are key enzymes in the synthesis of cell wall components. These monomeric enzymes belong to the 2-oxoglutarate dependent superfamily of enzymes characterized by a conserved jelly-roll framework. This algal P4H has high sequence similarity to the catalytic domain of the vertebrate, tetrameric collagen P4Hs, whereas there are distinct sequence differences with the oxygen-sensing hypoxia-inducible factor P4H subfamily of enzymes. We present here a 1.98-Å crystal structure of the algal Chlamydomonas reinhardtii P4H-1 complexed with Zn 2؉ and a proline-rich (SerPro) 5 substrate. This ternary complex captures the competent mode of binding of the peptide substrate, being bound in a lefthanded (poly)L-proline type II conformation in a tunnel shaped by two loops. These two loops are mostly disordered in the absence of the substrate. The importance of these loops for the function is confirmed by extensive mutagenesis, followed up by enzyme kinetic characterizations. These loops cover the central Ser-Pro-Ser tripeptide of the substrate such that the hydroxylation occurs in a highly buried space. This novel mode of binding does not depend on stacking interactions of the proline side chains with aromatic residues. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr 140 , as induced by the hydroxylation of the proline residue. The importance of these findings for understanding the specific binding and hydroxylation of (X-Pro-Gly) n sequences by collagen P4Hs is also discussed.

Structural and Functional Insights into Saccharomyces cerevisiae Tpa1, a Putative Prolylhydroxylase Influencing Translation Termination and Transcription

“…Similar conformational changes occur upon peptide binding for the corresponding ␤-hairpin of algal prolyl-4-hydroxylase (53). Whether rearrangements of the Tpa1 ␤-hairpin take place upon substrate binding is hard to predict in the absence of any information on Tpa1 substrates.…”

“…1E). The conservation of both folding and catalytically important residues between Tpa1 and prolyl-4-hydrolases (such as human PHD2 and algal prolyl-4-hydroxylase (44,53) Fig. 1C) strongly suggests that Tpa1 possesses a prolyl-4-hydroxylase enzymatic activity.…”

Bacillus anthracis Prolyl 4-Hydroxylase Modifies Collagen-like Substrates in Asymmetric Patterns

“…The model shows that the (P-P-G) 5 peptide forms an elongated PPII helical conformation similar to that observed in CrP4H (34,35) (Fig. 9A).…”

“…These are HIF␣-PHD2 (70), Pseudomonas aeruginosa PHD (PPHD) (37), CrP4H (34,35), vCPH (30), and apo-BaP4H (36). The crystal structures of BaP4H with bound cofactors display structural features characteristic of Fe(II)/␣KG-dependent enzymes consisting of the 2His-1carboxylate iron-binding motif.…”

“…A structure of an algal P4H from Chlamydomonas reinhardtii (CrP4H) complexed with a (S-P) 5 peptide is one of the few P4H crystal structures solved to date with a substrate bound in the active site (33). The structure illustrates various enzyme-substrate interactions in the binding groove and in the substrate binding loop (SBL) region adjacent to the active site, with the peptide adopting the PPII helix conformation (34,35). These highly mobile SBL regions are conserved through P4Hs and in the absence of a substrate one of the loops is usually disordered (30,36,37).…”