Structural basis for arginine glycosylation of host substrates by bacterial effector proteins

Jun Bae Park,Samuel Walpole,Jin Won Cho,Juyeon Kim,Sung-Hoon Jun,Ana A García-García,Philip R Hardwidge,Ramon Hurtado-Guerrero,Youngki Yoo,Samir El Qaidi,Miaomiao Wu,Hyun-Soo Cho,Young Hun Kim,Jesus Angulo,Jeon-Soo Shin,Serena Monaco,Michael P Hays

doi:10.1038/s41467-018-06680-6

Abstract

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

Highlights

The bacterial effector proteins SseK and non-locus of enterocyte effacement (LEE) encoded effector protein B (NleB) glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection
We demonstrate that the HLH domain is relevant to protein substrate recognition and the HEN residues are critical for catalysis
We investigated by NMR spectroscopy the glycosidic bond configuration of a GlcNAc-GAPDH187-203 glycopeptide, which was formed enzymatically by incubation with SseK1 and uridine 5′-diphosphate (UDP)-GlcNAc/MnCl2

Summary

Introduction

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Within proteins as acceptor substrates for GTs, the most prevalent glycosylated amino acids are serine and threonine (O-linked glycosylation), and asparagine (N-linked glycosylation) Another type of glycosylation was recently reported from studies of bacterial virulence proteins[5,6,7]. NleB target proteins include the tumor necrosis factor receptor type 1-associated death domain (TRADD), Fas-associated death domain (FADD), receptor-interacting serine/threonine-protein kinase 1 death domain (RIP1-DD), tumor necrosis factor receptor death domain (TNFR-DD), and GAPDH5–7. The T3SS effectors SseK1 and SseK2 from Salmonella typhimurium SL1344 are NleB orthologs that behave as NleB1-like GTs, they differ in protein substrate specificity[6,18]. Molecular dynamics simulations reveal that the presence of GlcNAc in the donor site induces conformational changes on the side chains of the peptide substrate so that the final arginine acceptor becomes properly oriented for a front face attack to the anomeric C1 carbon of the sugar

Methods

Results

Conclusion