Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis

Abstract

Glycogenin initiates the synthesis of a maltosaccharide chain covalently attached to itself on Tyr195 via a stepwise glucosylation reaction, priming glycogen synthesis. We have captured crystallographic snapshots of human glycogenin during its reaction cycle, revealing a dynamic conformational switch between ground and active states mediated by the sugar donor UDP-glucose. This switch includes the ordering of a polypeptide stretch containing Tyr195, and major movement of an approximately 30-residue "lid" segment covering the active site. The rearranged lid guides the nascent maltosaccharide chain into the active site in either an intra-or intersubunit mode dependent upon chain length and steric factors and positions the donor and acceptor sugar groups for catalysis. The Thr83Met mutation, which causes glycogen storage disease XV, is conformationally locked in the ground state and catalytically inactive. Our data highlight the conformational plasticity of glycogenin and coexistence of two modes of glucosylation as integral to its catalytic mechanism.glycogen storage disorder | glycosyltransferase

Discovery of Streptococcus pneumoniae Serotype 6 Variants with Glycosyltransferases Synthesizing Two Differing Repeating Units

“…Because residue 150 is variable, residues 149 -151 are herein named as the NXG motif to simplify its description. Crystallographic studies found the NXG motif to form a part of the ligand-binding pocket: NXG of Neisseria meningitidis LgtC surrounds the "C1" of the donor ligand (37), and the NXG of human glycogenin-1 interacts with the hydroxyl groups of "C2" and "C3" of Glc (32). Molecular modeling of WciN␣ using PHYRE2 (38) also predicted the NXG motif to form a ligand-binding pocket.…”

“…In contrast, the mutation at residue 150 is present in both German strains and is located within a highly conserved region. For instance, residues 148 -152 of WciN␣ are conserved in human glycogenin-1, except for residue 150 (FNAGV versus FNSGV) (32). Because residue 150 is variable, residues 149 -151 are herein named as the NXG motif to simplify its description.…”

“…MBO172 has WciN␣ (A150T), MBO182 has WciN␣ (D38N and A150T), MBO184 has WciN␣ (D38N), and MBO177 has WciN␣ (A150S). A150S variant was made because it is found in WciN␣ of serotype 33B and human glycogenin-1 (32 …”

Structural basis for the recruitment of glycogen synthase by glycogenin

“…Glycogenin is a member of the GT8 family of glycosyltransferases with a GT-A architecture containing an N-terminal catalytic domain with a single Rossmann fold that operates as an obligate dimer (12)(13)(14). The core catalytic domain is followed by a C-terminal extension of variable length and undefined structure.…”

High-Throughput Mass Spectrometry Applied to Structural Genomics

“…Histidine tag-associated modifications such as gluconoylation will also be lost following TEV cleavage. Intact mass measurements can be used to monitor deliberate modifications of the protein, such as biotinylation [26], reductive methylation [27], glycosylation [28], incorporation of SeMet or isotopically-labelled amino acids, or the removal of the tag by TEV cleavage. In all cases where a mass discrepancy cannot be unequivocally accounted for, the expression clone is (re)-sequenced.…”