Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a poorly characterized endoplasmic reticulum (ER)-localized transmembrane prolyl 4-hydroxylase (P4H-TM) is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome. Here, the 3D structure of the soluble human P4H-TM was solved using X-ray crystallography. The structure revealed an EF-domain with two Ca²⁺-binding motifs inserted to the catalytic domain. A substrate-binding cavity was formed between the EF-domain and the catalytic domain. The active site contained bound Fe²⁺ and N-oxalylglycine. Comparison to homologous structures complexed with peptide substrates showed that the substrate interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM. Differences to homologs were found in the extensive loop structures that surround the substrate-binding cavity and generate a negative surface charge. Ca²⁺-binding affinity of P4H-TM was determined to be within the range of physiological Ca²⁺concentration in the ER. The proximity of the EF-domain to the active site suggests that Ca²⁺-binding is relevant to the catalytic activity. P4H-TM was found both as a monomer and a dimer in solution, but the monomer-dimer equilibrium was not regulated by Ca²⁺. The solved 3D structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with the known P4Hs while possessing a unique property among the 2-oxoglutarate-dependent dioxygenases having an EF-domain and a catalytic activity potentially regulated by Ca²⁺.

中文翻译：

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跨膜脯氨酰4-羟化酶的结构揭示了EF和双加氧酶结构域的独特组织

脯氨酰4-羟化酶（P4Hs）催化肽基脯氨酸残基的翻译后羟化反应。除了胶原蛋白P4Hs和缺氧诱导因子P4Hs外，在动物中还发现了表征欠佳的内质网（ER）定位的跨膜脯氨酰4-羟化酶（P4H-TM）。P4H-TM变体与熟悉的神经系统HIDEA综合征相关。在这里，使用X射线晶体学解析了可溶性人P4H-TM的3D结构。该结构揭示了带有两个Ca ²⁺结合基序插入催化域的EF域。在EF-结构域和催化结构域之间形成底物结合腔。活性部位含有结合的Fe ²⁺和N-草酰甘氨酸。与与肽底物复合的同源结构的比较表明，底物相互作用残基和折叠在底物上的盖结构在P4H-TM中是保守的。在围绕底物结合腔并产生负表面电荷的广泛环结构中发现了与同源物的差异。确定P4H-TM的Ca ²⁺结合亲和力在ER中的生理Ca ²⁺浓度范围内。EF-结构域与活性位点的接近表明，Ca ²⁺结合与催化活性有关。发现P4H-TM既是单体又是溶液中的二聚体，但单体-二聚体平衡不受Ca ^2+的调节。解决的3D结构表明HIDEA变体导致P4H-TM功能丧失。总之，P4H-TM与已知的P4Hs共享关键的结构元素，同时在具有EF域和可能受Ca ²⁺调节的催化活性的2-氧代戊二酸依赖性双加氧酶中具有独特的特性。