Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3

Abstract

The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.

An intra-bacterial activity for a T3SS effector

“…Arginine glycosylation is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles. The NleB/SseK orthologs share high degrees of structural similarity and consist of a catalytic domain including essential DXD and HEN motifs, a helix-loop-helix (HLH) domain, and a C-terminal lid domain [4][5][6] . Several 'death domain'-containing proteins such as the Fas-Associated protein with Death Domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), and the receptor interaction serine/threonine-protein kinase 1 (RIPK1) are NleB/SseK substrates 2 .…”

The Long-wavelength Macromolecular Crystallography I23 at Diamond Light Source

“…Several novel structures have been solved based on the long wavelength data collected at I23 over the past two years. [28][29][30][31][32][33] The first published structure was that of ThcOx, an oxidase protein from the cyanobactin pathway. 34 Fig.3c.…”

Salmonellaeffectors SseK1 and SseK3 target death domain proteins in the TNF and TRAIL signaling pathways

“…While this work was in progress, the structure of SseK3 crystallized under somewhat different conditions and in a different space group was published [39]. Comparison of these structures and the recently deposited structure of NleB2 (PDB code 5H5Y) shows flexibility of the ~10 C-terminal residues, particularly in the absence of UDP.…”