Abstract
Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.
Highlights
Out of the four major classes of biological macromolecules: nucleic acids, proteins, lipids and carbohydrates, the latter is arguably the most complex and least understood
By performing domain prediction and pro le–pro le alignments with sequence pro les of proteins from Pedobacter heparinus (PDB), COG, Pfam and SCOP, we have previously shown that DS-epi[1] has no domain similarity to the alginate and heparan sulfate epimerases but is remotely related to polysaccharide-degrading bacterial enzymes.[19]
An experimentally supported model of the full-length Golgi luminal DS-epi[1] was created, where 80% of the 958 amino acidprotein was solved by macromolecular crystallography and the rest using targeted cross-linking mass spectrometry (TX-MS) experiments and Rosetta modeling
Summary
Out of the four major classes of biological macromolecules: nucleic acids, proteins, lipids and carbohydrates, the latter is arguably the most complex and least understood. One of the most common GAG types, chondroitin/dermatan sulfate (CS/DS), is built up by repeating disaccharide units of D-glucuronic acid (GlcA) and N-acetyl-Dgalactosamine (GalNAc). The CS/DS polymer can be modi ed by epimerization and sulfation, introducing the potential for vast structural diversity (Fig. 1).[4] The two human dermatan sulfate epimerases DS-epi[1] and DS-epi[2], essential for the epimerization of position 5 of GlcA residues to form L-iduronic acid (IdoA), were previously identi ed, cloned, expressed, puri ed and functionally evaluated by our group.[5,6]
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