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

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 S_Ni mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

Fig. 1

SseK1 is a retaining-glycosyltransferase. NMR spectra showing the reaction product of GAPDH_195-203 with SseK1. a Decoupled ¹H-¹³C HSQC spectrum (800 MHz) showing the anomeric region, highlighting the presence of α-D-GlcNAc-GAPDH_187-203, with a large ¹³C upfield shift relative to the free species. b Expansion of ¹H-¹³C CLIP HSQC spectrum (500 MHz) with no decoupling to measure the anomeric ¹J_CH coupling in α-GlcNAc-GAPDH_187-203. A value of 169 Hz indicates an α-configuration. c ¹H-¹H TOCSY spectrum highlighting through-bond correlation between the anomeric proton of α-D-GlcNAc in GAPDH_187-203-α-GlcNAc and an arginine η-proton

Fig. 2

Overall enzyme architecture. a Colored ovals show the full-length amino acid sequence and the black line under the oval shows the amino acid sequence used for overexpression and crystallization. b Arrange crystal structure and electron density maps of each ligands (2Fo – Fc electron density maps of UDP on SseK1 and UDP & L-arginine on SseK2 contoured at 1σ and UDP and the others are 2σ). c Numbering of the α-helices and β-strands of SseK2 and d superimposition of SseK1, SseK2, and NleB2. e Each sub-domain is presented in different colors (blue: HLH domain, yellow: catalytic domain, red: lid domain)

Fig. 3

UDP-GlcNAc binding mode in SseK2. a Uracil moiety of UDP-GlcNAc interacts with SseK2 through hydrogen bonds and π-π stacking (top panel), but SseK1 uses a slightly different mechanism (second and third panel). Uracil binding mode of SseK3 is similar to SseK2 instead of SseK1 (bottom panel). b GlcNAc moiety of UDP-GlcNAc interacts with Asp204, Arg207, Asp239, and Arg348 by hydrogen bonds. The carbonyl group of the acetyl of GlcNAc interacts with a water molecule to stabilize the divalent metal ion. c Manganese ion coordinates six oxygens from pyrophosphate, Ser340, Asn338, Asp241, and water. The DxD motif stabilizes both UDP-GlcNAc and manganese ion. *Amino acid numbering in brackets refers to conserved sequence of SseK1. Black dashed lines represent hydrogen bonds

Fig. 4

Donor substrate-mediated conformational changes. a Comparison of surface structures between apo form and UDP-GlcNAc bound SseK2. The flexible C-terminus creates an open conformation for access of UDP-GlcNAc and after binding of the UDP-GlcNAc, the C-terminal lid domain (red color) interacts with UDP-GlcNAc and is fixed in closed conformation. Yellow stick represents UDP-GlcNAc. b After the donor-substrate binds to the active site, the α10 helix tilts about 3.5 degrees and the flexible C-terminal lid domain is fixed towards the UDP-GlcNAc. Due to hydrogen bonding at Arg207, Asp204, and aromatic stacking at Phe203, the α10 helix is able to tilt. The flexible C-terminal lid domain can be fixed by hydrogen bonding of Ser340, Ser345, Ser346, Arg348, and Trp347. Orange and blue-green colors represent SseK2 apo and UDP-GlcNAc bound SseK2 structure, respectively. Black dashed lines represent hydrogen bonds. c Direction of the lid-domain is different between the SseK1 (in green) and SseK2 (in white) structures. Curved arrows (in black) each correspond to the direction of the backbone of the lid-domain

Fig. 5

Binding modes of short peptide substrates. Binding epitope mappings of a FADD_110-118 b TRADD_229-237 and c, d GAPDH_195-203 peptides in the presence of 25 μM SseK1. Samples in a, b and d contained 25 μM Mn²⁺, and 25 μM UDP. All STD intensities normalized against Hζ2 of the tryptophan. Colored circles represent magnitude of normalized intensities (blue:< 40%, pink: 40–70%, red:> 70%). Comparison of GAPDH_195–203 binding to SseK1, c in the absence, and, d in the presence of Mn²⁺ and UDP, reveals a significant change in the binding mode of the substrate peptide upon addition of the cofactor and the nucleotide diphosphate. For STD NMR study of binding to SseK2 see Supplementary Fig. 5

Fig. 6

HEN motif plays a key role in NleB/SseK enzyme activity. a SseK1 mutants were generated and the cellular function in HEK293T cells was investigated. A non-reducing gel (right panel) was used to confirm the presence of the TRADD oligomer. Mutants in red represent mutations of residues proposed to be catalytically important. Data represent at least three repetitions. b The NF-κB level in A549-NF-κB luc cells was measured to check the enzymatic functions. Data represent the mean and standard deviation in triplicate. Multiple comparisons perform by one-way ANOVA followed by Turkey’s Multiple Comparison Test (**P < 0.01, ***P < 0.001 compare to WT). c Enzyme kinetic assays of SseK1 and SseK2, respectively. d In vitro glycosylation of FADD by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). e In vitro glycosylation of TRADD by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). f In vitro glycosylation of GAPDH by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). g Glycosylation of TRADD after co-transfection with either NleB1 or SseK1 (WT and HEN mutants) in HEK293T cells. FLAG-TRADD was immunoprecipitated and then immunoblotted using an anti-Arg-GlcNAc antibody. h Glycosylation of GAPDH after co-transfection with either NleB1 or SseK1 (WT and HEN mutants) in HEK293T cells. FLAG-TRADD was immunoprecipitated and then immunoblotted using an anti-Arg-GlcNAc antibody. i Colonization (log10 CFUs/g colon) of indicated C. rodentium strains (14 days post-gavage) in C57BL/6 J mice (n = 6). Asterisks indicate significantly different colonization magnitude as compared to WT; Kruskal-Wallis test. Uncropped blots are shown in Supplementary Figs. 21 and 22