Structure of transmembrane prolyl 4-hydroxylase reveals unique organization of EF and dioxygenase domains

Abstract

Edited by Joseph Jez Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H-the poorly characterized endoplasmic reticulum-localized transmembrane prolyl 4-hydroxylase (P4H-TM)-is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF domain with two Ca 2+-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF domain and the conserved core of the catalytic domain. The proximity of the EF domain to the active site suggests that Ca 2+ binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca 2+-binding affinity of P4H-TM is within the range of physiological Ca 2+ concentration in the endoplasmic reticulum. P4H-TM was found both as a monomer and a dimer in the solution, but the monomer-dimer equilibrium was not regulated by Ca 2+. The catalytic site contained bound Fe 2+ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison with homologous P4H structures complexed with peptide substrates showed that the substrateinteracting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having a unique EF domain. This article contains supporting information.

HIDEA syndrome is caused by biallelic, pathogenic, rare or founder P4HTM variants impacting the active site or the overall stability of the P4H‐TM protein

“…We characterized the HIDEA causing variants using the recently published multiple sequence alignments and crystal structure showing the residues from 107 to 481 of the prevalent 502‐residue form of P4H‐TM (Figure 2A ). 10 Nonsense, frameshift, and in‐frame deletion variants in P4HTM (Figure 2B–G ) are likely be degraded by nonsense‐mediated decay, or degraded due to protein misfolding and will not retain any P4H‐TM enzyme activity. The P4HTM missense variants p.(Thr361Ile) and p.(Pro413Leu) are residues conserved in P4Hs, while p.(Glu312Lys) is conserved in collagen prolyl 4‐hydroxylases but not in the hypoxia‐inducible factor prolyl 4‐hydroxylases.…”

P4HTM: A Novel Downstream Target of GATA3 in Breast Cancer

“…P4HTM, a member of the P4H family, encodes an ER transmembrane domain near its N-terminus and catalyzes the hydroxylation of proline residues (24,25). In addition to HIF1a, collagen, bronectin, and laminin have been identi ed as P4HTM-mediated proline hydroxylation substrates.…”

LcSAO1, an Unconventional DOXB Clade 2OGD Enzyme from Ligusticum chuanxiong Catalyzes the Biosynthesis of Plant-Derived Natural Medicine Butylphthalide

“…The structure obtained by AlphaFold2 was then optimized using FastRelax module of multi-functional protein design platform Rosetta to eliminate irrational conformations in the structure [33]. The structure of LcSAO1 is highly similar to the three-dimensional structure of DOXB family proteins, whose crystal structure has been resolved [34], which implies its function of hydroxylation (Figure S6). Next, senkyunolide A was docked into the active site of LcSAO1 using RosettaScripts with Ligand_Docking, followed by conformation optimizing by FastRelax [33].…”