In a study published in Molecular Cell, researchers from the Institute of Biophysics of Chinese Academy of Sciences, National Institute of Biological Sciences, and Huazhong Agricultural University deciphered the precise molecular mechanism of blocking host death receptor signaling by Enteropathogenic E. coli (EPEC) arginine GlcNAcyltransferase NleB.

Employing specialized effectors to manipulate or block host signaling pathways is a common strategy used by bacterial pathogen. Death receptor signaling is an emerging strategy for host counteracting bacterial infection through stimulation of NF-κB-mediated transcriptional response for inflammation and activation of caspase-mediated programmed apoptosis.

A type III secretion system effector of EPEC, NleB, has been identified. It catalyzes the attachment of N-acetylglucosamine (GlcNAc) onto a crucial arginine of the death domain (DD) and there by disrupts death-domain interactions. NleB-catalyzed arginine GlcNAcylation represents an unprecedented post-translation modification (PTM) in host cells. However, the mechanism underlying NleB recognition of death-domain substrates and catalysis of arginine GlcNAcylation is unknown.

The crystal structures of NleB alone, NleB in complex with death-domain substrate (FADD-DD) and the sugar donor, as well as arginine-GlcNAcylated death domains (TRADD-DD and RIPK1-DD) were determined in this study, which provided structural snapshots for all major steps in NleB-catalyzed arginine GlcNAcylation of death domains. The structural data, together with in vitro biochemical analyses and in vivo mouse infection assays, revealed the detailed molecular mechanism for NleB functioning.

Researchers then found that extensive hydrogen-bond and electrostatic interactions at the intermolecular interface confer NleB with specific recognition and high affinity to a subgroup of death-domain substrates. The side chain of acceptor arginine stretches into a narrow cleft in NleB, generating a bidentate hydrogen-bond interaction between the arginine guanidinium and the carboxyl group of Glu253 of NleB. Glu253 acts as the base to deprotonate the guanidinium in the arginine substrate.

The arginine nucleophilically attacks the C1 atom of UDP-GlcNAc, forming an oxocarbenium ion-like transition state that progresses to SN2-like displacement of the leaving group (UDP).

NleB employs an inverting sugar-transfer mechanism to convert α-anomeric configuration of C1 in UDP-GlcNAc intoa β-glycosidic linkage of GlcNAcylated arginine. It is a broad-spectrum inhibitor of death receptor signaling due to its pleotropic blocking of death-domain interactions. NleB-catalyzed arginine GlcNAcylation, as a novel type of PTM, is quite different from other known glycosylation in host cells.

The finding provided a comprehensive and in-depth understanding of the enzymatic reaction mechanism of arginine GlcNAcylation.

This study expanded the knowledge about bacterial effectors manipulation of host signaling pathways via novel PTM, and will benefit NleB-based drug development for antagonizing EPEC infection.