Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

• Published in: Nature communications Vol. 10; no. 1; pp. 4910 - 16
• Main Authors: Pfeffer, Inga, Brewitz, Lennart, Krojer, Tobias, Jensen, Sacha A., Kochan, Grazyna T., Kershaw, Nadia J., Hewitson, Kirsty S., McNeill, Luke A., Kramer, Holger, Münzel, Martin, Hopkinson, Richard J., Oppermann, Udo, Handford, Penny A., McDonough, Michael A., Schofield, Christopher J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.10.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 631/45; 631/535; 631/92; 82/58; 82/80; 82/83; Amino Acid Sequence; Asparagine; Asparagine - metabolism; Binding; Calcium; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Catalytic Domain; Crystal structure; Crystallography; Disulfides - chemistry; Disulfides - metabolism; Domains; Endoplasmic reticulum; Epidermal growth factor; Epidermal Growth Factor - metabolism; Ferrous Compounds - chemistry; Ferrous Compounds - metabolism; Growth factors; Humanities and Social Sciences; Humans; Hydroxylase; Hydroxylation; Iron; Ketoglutaric acid; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; multidisciplinary; Muscle Proteins - chemistry; Muscle Proteins - genetics; Muscle Proteins - metabolism; Organic chemistry; Oxygenase; Protein Conformation; Science; Science (multidisciplinary); Substrates
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-12711-7

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.