NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machinery

NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machinery† Ana Garc´ ıa-Garc´ ıa, a Thomas Hicks, b Samir El Qaidi, c Congrui Zhu, c Philip R. Hardwidge, c Jes´ us Angulo * bde and Ramon Hurtado-Guerrero * afg NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conserved Citrobacter rodentium NleB and E. coli NleB1 proteins display a broad selectivity towards host proteins, Salmonella enterica SseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284 NleB/NleB1 , a second-shell residue contiguous to the catalytic machinery. Tyr284 NleB/NleB1 is important in coupling protein substrate binding to catalysis. This is exemplified by S286Y SseK1 and N302Y SseK2 mutants, which become active towards FADD and DR3 death domains, respectively, and whose kinetic properties match those of enterohemorrhagic E. coli NleB1. The integration of these mutants into S. enterica increases S. enterica survival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Our findings provide insights into how these enzymes finely tune arginine-glycosylation and, in turn, bacterial virulence. In addition, our data show how promiscuous glycosyltransferases preferentially glycosylate specific protein substrates. Introduction Glycosyltransferases (GTs) are ubiquitous enzymes that transfer a sugar moiety from either sugar nucleotides or lipid phosphate sugars to small molecules, lipids, carbohydrates, small RNAs, and proteins. 1–3 Glycosylation of proteins is generally a multistep process in most eukaryotic organisms, 3 and likely the most abundant posttranslational modication (PTM), at least in humans. 3 Glycans are generally attached to proteins in four different ways –N-linked to asparagine (Asn), O-linked to the hydroxyl groups of serine (Ser) threonine (Thr), tyrosine (Tyr), or C-linked to tryptophan (Trp) and glypiation. 3 Each type of protein glycosylation is initiated by one or more unique protein GTs that in turn dene the different glycosylation pathways. While most of these pathways occur post-translationally, initiation of N-glycosylation and likely POMT-directed O-man- nosylation occur co-translationally. 3,4 A rare PTM described a few years ago, arginine-glycosylation, is catalyzed by Gram-negative bacterial GTs. 5 This unusual PTM occurs on the arginine guanidinium group, a very poor nucleophile. In Pseudomonas and Neisseria species, arginineglycosylation is catalyzed by EarP, while in enteropathogens, it is catalyzed by the type III secretion system effectors arginine GTs NleB and SseK. 6–9 EarP is a rhamnosyl-transferase that uniquely glycosylates the bacterial translation elongation factor P (EF-P) to activate its function and drive bacterial pathogenicity. 6 The NleB/SseK GTs transfer GlcNAc to arginines of several mammalian proteins and to at least ve bacterial proteins. 7–12 The NleB/SseK GTs are not classied in the CAZy database (GTnc). 13 While C. rodentium only encodes one NleB, most E. coli strains encode two NleB proteins named NleB1 and NleB2. For several years, the role of NleB2 was unclear 10 until a recent publication reported that NleB2 is an arginine GT that preferably transfers glucose to RIPK1, inhibiting host protein function similarly to other NleB/SseK GTs. 14 The change in sugar donor preference was attributed to Ser252 NleB2 , which corresponds to a Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Mariano Esquillor s/n, Campus Rio Ebro, Edicio I+D, Zaragoza, Spain. E-mail: rhurtado@bi.es b School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK c College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA d Departamento de Qu´ ımica Org´ anica, Universidad de Sevilla, Sevilla, 41012, Spain. E-mail: [email protected] e Instituto de Investigaciones Qu´ ımicas (CSIC-US), Sevilla, 41092, Spain f Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, School of Dentistry, University of Copenhagen, Copenhagen, Denmark g Fundaci´ on ARAID, Zaragoza, Spain †Electronic supplementary information (ESI) available: Supporting methodology, 9 supporting gures and 5 supporting tables. See DOI: 10.1039/d1sc04065k Cite this: Chem. Sci.,2021,12,12181 All publication charges for this article have been paid for by the Royal Society of Chemistry Received 25th July 2021 Accepted 12th August 2021 DOI: 10.1039/d1sc04065k rsc.li/chemical-science © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,12181–12191 | 12181 Chemical Science EDGE ARTICLE Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue Fig. 1 Analysis of the interacting residues in the NleB1 EPEC –FADD DD interface. (a) Multiple sequence alignment of CrNleB, NleB1 EPEC , NleB1 EHEC , SseK1 wt , SseK2 wt and SseK3 wt . Residues are color-coded by their degree of sequence conservation where black, grey and white colors denote identity, high similarity and dissimilarity, respectively. Shown above the NleB1 EPEC sequence, in gray (catalytic domain) and brown (HLH domain), are the secondary structure elements (a-helices and b-strands) based on the NleB1 EPEC –UDP–Mn 2+ –FADD DD structure (PDB entry 6ACI 19 ). The residues forming part of the C-terminal lid are indicated within a red box while a blue rectangle determines the five C-terminal residues. The five inverted green triangles indicate the residues in NleB GTs that are non-conserved or partly conserved with the SseK GTs and are engaged in FADD DD interaction. These residues were targeted for site-directed mutagenesis in SseK1 wt and SseK2 wt . (b) Cartoon representation of the NleB1 EPEC –UDP–Mn 2+ –FADD DD . The catalytic and HLH domains of NleB1 EPEC are shown in gray and brown, respectively. The FADD DD is shown in cyan. Residues are shown as sticks with carbon atoms with the corresponding colors indicated above. UDP and Mn 2+ are shown as green carbon atoms and as a pink sphere, respectively; hydrogen bond interactions are shown as dotted orange lines but only for residues interacting at the interface of the complex. The interactions with UDP and Mn 2+ have been extensively discussed before 16,17 and will not be further discussed. 12182 |Chem. Sci.,2021,12,12181–12191 © 2021 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online a Gly residue in all homologous sequences. 14 CrNleB is highly conserved in the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) NleB1s. 15 Particularly, the identity between these enzymes is 89% between CrNleB and NleB1s, and 98% between NleB1s (Fig. 1a). Salmonella enterica strains encode up to three functional NleB orthologs named SseK1, SseK2, and SseK3. When the CrNleB/NleB1s EPEC/EHEC are compared to SseK1/2/3, the sequence identities drop signicantly, ranging from 51 to 57% (Fig. 1a). At the structural level, these enzymes show a high degree of similarity and are built by two conserved major domains and a C-terminal lid, which is also required for the catalytic activity of the enzyme. The GT-A fold-adopting catalytic domain is the largest domain and includes the essential DxD and HEN (His–Glu–Asn) motifs. The helix–loop–helix (HLH) domain comprises two helices, a3 and a4, connected by a loop 16–19 (Fig. 1a and b). NleB GlcNAc-transferase activity is essential to bacterial virulence. 7 Multiple host protein substrates for the CrNleB and NleB1 EPEC/EHEC have been described and include the death domains (DD) of tumor necrosis factor receptor 1 (TNFR1), the TNFR1-associated death domain protein (TRADD), the receptorinteracting serine/threonine-protein kinase 1 (RIPK1), the TNF- receptor superfamily member 25 (DR3 or TNFRSF25), and FAS- associated death domain protein (FADD). 9,19,20 In addition, these enzymes glycosylate other proteins non-containing DDs. CrNleB and NleB1 EHEC , but not NleB1 EPEC , glycosylate GAPDH, 10 and NleB1 EPEC glycosylates HIF-1a. 21 Overall, NleB1 disrupts TNFR-associated factor (TRAF) signaling, leading to inhibition of the pro-inammatory NF-kB pathway. 7,9,10 SseK1, SseK2, and SseK3 have a narrower protein substrate selectivity; SseK1 glycosylates TRADD 9 and GAPDH, 10 but not FADD; 10 SseK2 glycosylates FADD 10 but not TRADD 17 or GAPDH; 10 SseK3 glycosylates TNRF1, 18 TRAIL, 18 and the small GTPase Rab1, 22 but not GAPDH 10 or FADD. 10 This illustrates that although the SseK and NleB GTs are highly similar at the sequence and structural level, they display dissimilarities in their protein substrate selectivity. Furthermore, the SseK GTs are inactive towards some NleB-specic protein substrates. 10,17 To address the molecular basis of NleB/SseK protein substrate selectivity and to determine why some SseK GTs do not glycosylate particular protein substrates, we report herein a multidisciplinary approach on wild type (wt) NleB1 EHEC , SseK1, and SseK2, combined with the characterization of different SseK1 and SseK2 mutants, which reveals that a single second-shell residue near to the catalytic machinery, nely tunes substrate selectivity and catalysis. We also show that optimal kinetic parameters are accomplished by the mutants S286Y SseK1 and N302Y SseK2 , which contain a Tyr residue that replaces SseK1 wt Ser286 and SseK2 wt Asn302. We recover full activity with S286Y SseK1 on FADD DD and N302Y SseK2 on DR3 DD . Finally, we demonstrate that the integration of S286Y SseK1 and N302Y SseK2 mutants in a Salmonella enterica strain devoid of all SseK enzymes increases Salmonella survival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Results and discussion Bioinformatics and structural analysis of NleB/SseK GTs To determine the molecular basis of why SseK1 wt does not glycosylate FADD DD , we performed a multiple alignment analysis of CrNleB, NleB1 EPEC/EHEC , and SseK GTs, and also carefully inspected the residues of NleB1 EPEC engaged in recognition of FADD DD (Fig. 1a and b; PDB entry 6ACI 19 ). Fourteen NleB1 EPEC residues established diverse types of interactions with FADD DD residues, with salt bridge interactions being the most prevalent, followed by hydrogen bond/hydrophobic interactions (Fig. 1b and Table S1†). Five of these 14 residues were in the HLH domain (Tyr145, Glu149, Asp151, Tyr153 and Leu154), while the others (Glu253, Lys277, Asp279, Tyr283, Tyr284, Asp285, Lys289, Lys292, and Tyr303) were located in the catalytic domain (Fig. 1b and Table S1†). Interestingly, not a single residue of the C-terminal lid interacted with FADD DD residues. In addition, but with the exception of Glu149, which interacted through its backbone with the Arg135 FADD side chain, the other residues interacted with FADD residues through their side chains. A smaller number of FADD DD residues were engaged in recognition of NleB1 EPEC . All these nine residues were located along a2, a3, a4, and the loops connecting them (Fig. S1 and Table S1†). Again, and as found for NleB1 EPEC residues, most of the FADD DD residues involved in recognition of NleB1 EPEC utilized their side chains. Four of these nine residues (Trp112 FADD , Arg117 FADD , Val121 FADD , and Ile126 FADD ) were completely conserved between different DDs and likely comprise the minimal epitope for NleB/SseK GTs recognition, as suggested before. 17,19 Note that Arg117 FADD is the acceptor site and is engaged in a salt bridge with the proposed catalytic base Glu253 (ref. 19) (Fig. 1b). A thorough comparison of the grade of conservation of the NleB1 EPEC fourteen interacting residues with aligned residues from other orthologs indicated that only ve residues might be responsible for the null activity of SseK1 wt on FADD DD (Table S1†). These residues were mostly non-conserved or partially conserved with SseK1 wt residues. Tyr145 and Glu149 were quite close in the structure and located in a loop connecting a3 with a4 of the HLH domain (Fig. 1b). The other three residues, Tyr284, Lys289, and Lys292, were located in the catalytic domain and exclusively in a9 (Fig. 1a). While both Lys residues were proximal in the structure and established salt bridges with Asp123 FADD and Glu130 FADD , Tyr284 was more isolated from them and interacted through its side chain to Val121 FADD backbone and Ile126 FADD side chain (Fig. 1 and Table S1†). Tyr284 was also engaged in a CH–pinteraction with the proximal Tyr283 side chain, which likely controls the interaction between the acceptor Arg117 FADD and the catalytic base Glu253 (Fig. 1b). Hence, Tyr284 is a second-shell residue with respect to the catalytic machinery in which Glu253 is one of the key players. The S286Y SseK1 mutation switches on activity against FADD DD Based on the above analysis, we selected ve residues from NleB GTs to perform site-directed mutagenesis on the corresponding © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,12181–12191 | 12183 Edge Article Chemical Science Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online aligned residues in SseK1 wt and SseK2 wt . To simplify the number of mutations in SseK1 wt and to minimize the potential combinations among them, we established three groups of residues: the two pairs Tyr145–Glu149 NleB1 , and Lys289– Lys292 NleB1 , based on their proximity at structural level, and Tyr284 NleB1 as one unique and independent residue from the others. To initiate this study, we determined the kinetic parameters of NleB1 EHEC on UDP-GlcNAc and FADD DD (see Fig. 2a, le panel, ESI, Fig. S2, and Table S2†). NleB1 EHEC displayed a clear substrate inhibition prole under the presence of variable concentrations of FADD DD (Fig. 2a; K i ¼793 160 mM) that was not present in the other SseK2 wt and mutant proteins. This behavior might be attributed to a different non-productive FADD DD structural arrangement under high concentrations of this substrate. On the contrary, substrate inhibition under variable concentrations of UDP-GlcNAc was barely present (Fig. S2†). The K m s for UDP-GlcNAc and FADD DD were 125 33 and 13 2.5 mM, respectively, and the k cat was 100 min 1 (Fig. 2b, leand middle panels, and Table S2†), a value in Fig. 2 Enzyme kinetics and ITC experiments of NleB1 EHEC /SseK1 wt /SseK2 wt and mutants on FADD DD . (a) Glycosylation kinetics of NleB1 EHEC / SseK1 wt /SseK2 wt and mutants against FADD DD . (b) Plots comparing the K m ,k cat and catalytic efficiency (k cat /K m ) of the different NleB1 EHEC / SseK1 wt /SseK2 wt and mutants. Additional kinetic data are given in Table S2.†Asterisks indicate that the kinetic parameters for SseK2 wt are estimated due to its poorer binding to FADD DD . (c) (left) Thermodynamic dissection of the interaction of the different enzyme forms with FADD DD . The binding Gibbs energy (DG), enthalpy (DH), and entropy (TDS) are in kcal mol 1 . Any negative value represents a favorable contribution to the binding, whereas a positive value represents an unfavorable contribution (right) graph depicting the K d s of the different enzymes. 12184 |Chem. Sci.,2021,12,12181–12191 © 2021 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online agreement with other previous reported k cat values for a similar protein GT such as EarP 23 (k cat of 35 min 1 ), and an unrelated one such as PoFUT2 (ref. 24) (k cat of 144 min 1 ), both of which are glycosyltransferases that glycosylate other types of folded domains. Furthermore, these k cat values also match reported k cat values for unstructured peptides in the presence of other protein GTs (a range between 46–400 min 1 , and 18–300 min 1 for different peptides using GalNAc-T3 (ref. 25) and N-glycosyl- transferase, 26,27 respectively). As expected from previous 8,9 and our own studies, 10 SseK1 wt was inactive on FADD DD and SseK2 wt was slow on FADD DD (Fig. 2a, middle and right panel, and Table S2†). Particularly, the K m ,k cat , and catalytic efficiency of SseK2 wt were 95-, 6.7-, and 630-fold worse than those of NleB1 EHEC (Fig. 2b). Note that the kinetic parameters for SseK2 wt are estimated due to its poor binding to FADD DD . Seeking to switch on the activity of SseK1 wt towards FADD DD , we characterized the double mutants M147Y–K151E SseK1 and N291K–R294K SseK1 , and the single mutant S286Y SseK1 . In all of these mutants, we replaced the SseK1 wt residues by the corresponding positions in NleB1 EPEC/EHEC . While the initial velocity of the double mutants was very slow (46-fold worse than the NleB1 EHEC initial velocity at 800 mM FADD DD ; Fig. 2a, middle panel), strikingly, the k cat for the single mutant S286Y SseK1 matched that of NleB1 EHEC (only 1.38-fold worse; Fig. 2b, middle panel), implying that with a single mutation we reached the optimal k cat found for NleB1 EHEC . On the contrary, K m and the catalytic efficiency for S286Y SseK1 were slightly worse (4.4- and 6.7-fold worse than the ones reported for NleB1 EHEC ; Fig. 2b, leand right panels, and Table S2†). To obtain an SseK1 mutant with similar kinetic parameters as those of NleB1 EHEC , we combined the double mutants to generate a quadruple mutant (M147Y-K151E-N291K-R294K SseK1 ). We further added the S286Y SseK1 mutation to the latter mutant generating a quintuple mutant (M147Y-K151E-S286Y-N291K-R294K SseK1 ). Additionally, we made another mutant combining the quintuple mutant with a deletion of the C-terminal last 5 residues only found in SseK1 wt (Fig. 1a) named as quintuple-del mutant. The k cat ,K m and catalytic efficiency for the quadruple mutant were 4.3-/2.7-, 6.1-/1.4-, and 27-/4-fold worse than the ones for NleB1 EHEC and S286Y SseK1 , respectively (Fig. 2a, b, and Table S2†). This demonstrates that the kinetic parameters of the quadruple mutant are closer to those of S286Y SseK1 than those of NleB1 EHEC . Hence, either the combination of multiple changes (quadruple mutant) or just a single mutation (S286Y SseK1 mutant) leading to higher affinity of SseK1 towards FADD DD are two different approaches to achieve k cat values close to that of NleB1 EHEC . The kinetic parameters of the quintuple and quintuple-del, mainly k cat and k cat /K m , were highly similar to each other and very close to those of S286Y SseK1 (Fig. 2b, middle and right panels). However, they differed slightly more in their K m s, with the quintuple/quintuple-del mutants K m s being 2-fold lower than that of S286Y (Fig. 2b, lepanel). The reduction in K m for the quintuple/quintuple-del mutants enhanced their catalytic efficiencies, approximating their values to that of NleB1 EHEC . Overall, the removal of the C-terminal 5 amino acids in SseK1 wt and the addition of 5 mutations led to an SseK1 form with the closest kinetic parameters to those of NleB1 EHEC , mainly due to the lowest K m towards FADD DD of these SseK1 mutants. Having established that a single mutation (S286Y SseK1 ) was sufficient to achieve optimal kinetic parameters and render the best k cat of all the characterized mutants, we mutated Ser286 S- seK1 and Asn302 SseK2 to Asn/Ile, and Ser/Ile/Tyr, respectively, rendering the mutants S286N SseK1 , S286I SseK1 , N302S SseK2 , N302I SseK2 and N302Y SseK2 . These single mutants are derived from the alignment of Ser286 SseK1 with Asn302 SseK2 , Ile289 SseK3 and Tyr284 CrNleB/NleB1 (Fig. 1a). As expected from the previous results 10 of SseK3 wt on FADD DD , the S286I SseK1 and N302I SseK2 were completely inactive, and N302S SseK2 was also completely inactive on FADD DD , implying that both Ser or Ile residues are likely deleterious for catalysis (Fig. 2a, middle and right panel). In addition, S286N SseK1 showed poor glycosylation of FADD DD (15-fold worse initial velocity than the one reported for Nle- B1 EHEC at 800 mM FADD DD ) as found for SseK2 wt (Fig. 2a, middle panel). Although N302Y SseK2 achieved a highly similar k cat to those of NleB1 EHEC and S286Y SseK1 , its K m towards FADD DD was worse (3- and 12-fold higher than those of S286Y SseK1 and Nle- B1 EHEC , respectively). This caused a drop in catalytic efficiency compared to that of NleB1 EHEC that was more drastic than that for S286Y (3- and 20-fold worse to those of S286Y SseK1 and NleB1 EHEC , respectively; Fig. 2a, b, and Table S2†). Overall, our data indicate that a single mutation, either from Ser286 SseK1 or Asn302 SseK2 to Tyr, is sufficient to switch on and improve SseK1 and SseK2 glycosylation on FADD DD , respectively. This mutation allows reaching kinetic parameters very close to those of NleB1 EHEC . Binding of SseK1 to FADD DD is dramatically increased by the S286Y mutation To determine the thermodynamic parameters of the mutants to FADD DD , we performed isothermal titration calorimetry (ITC) experiments. First, we determined the K d of UDP for binding to NleB1 EHEC in the presence of MnCl 2 (K d ¼14.92 1.11 mM) (Table S3 and Fig. S3†). Then, we evaluated whether these enzymes require UDP binding prior to binding FADD DD . While in the absence of UDP, NleB1 EHEC or S286Y SseK1 did not show any binding to FADD DD , this turned out to be the opposite in the presence of excess UDP (Fig. 2c, Table S3 and Fig. S3†). This provides compelling evidence that the NleB/SseK GTs likely follow an ordered bi–bi kinetic mechanism. In this mechanism, these enzymes are likely in an inactive state in the apo form (open C-terminal lid) that only shis to the active state (closed C-terminal lid) in the presence of UDP-GlcNAc/MnCl 2 . This mechanism also implies an induced-t mechanism by UDP- GlcNAc, in which this sugar nucleotide induces the closure of the C-terminal lid leading to the active state (Fig. S4†). The induced-t mechanism has been also proposed for unrelated glycosyltransferases such as GalNAc-T2, B4GALT1 and the lactose synthase. 28–30 Once we determined that FADD DD binding to these enzymes requires prior UDP binding, we measured the thermodynamic parameters for all mutants versus FADD DD under an excess of UDP. We could only get titration for NleB1 EHEC and the © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,12181–12191 | 12185 Edge Article Chemical Science Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online S286Y SseK1 , N302Y SseK2 , quadruple, quintuple, and quintupledel mutants (Fig. S3†). Detailed analysis of the thermodynamic parameters of the interaction showed that the binding of FADD DD to NleB1 EHEC and the mutants was largely entropy-driven (TDS), while the binding of UDP was favored by a gain in enthalpy (DH), with a reduced entropic component (Fig. 2c and Table S3†), implying distinct interaction behaviors between these molecules. The unique thermodynamic prole exhibited by FADD DD might be due to the release of a vast number of surface water molecules from both FADD DD and NleB1 EHEC /SseK1/SseK2 mutants upon binding, promoting a favorable desolvation entropy. On the contrary, the signicant reduction in donor substrate mobility upon binding to the enzyme, along with the large number of hydrogen bonds between UDP or UDP-GlcNAc and these enzymes are largely the major factors explaining the reduction in the entropic component and the favorable enthalpy. 17,19 Interestingly, the single mutants S286Y SseK1 and N302Y SseK2 achieve favorable binding Gibbs energy to FADD by reducing the benecial entropy component of the interaction, what is accompanied by a more favorable enthalpy. Binding to FADD thus globally follows a pattern of enthalpy–entropy compensation where multiple mutants show similar thermodynamic proles to that of NleB1, with single mutants benetting from enthalpy, suggesting that the solvation/desolvation process at the interface of interaction with FADD is more similar to NleB1 for the multiple mutants than for the single mutants (Fig. S5†). The K d s are in the low mM range except for that of N302Y SseK2 . Although the K d s are much lower than the K m s, there is some correlation between the K m s and the K d s: those enzymes with lower K m values also possess lower K d s (Table S3†). Again, NleB1 EHEC displays the highest affinity (K d ¼0.2  0.04 mM), being 3-, 6-, 13-, and 472-fold better than those of quintuple-del, quintuple/S286Y SseK1 , quadruple, and N302Y SseK2 mutants, respectively (Fig. 2c, right panel, and Table S3†). Overall, our data show that the improvement in binding of SseK1 mutants and N302Y SseK2 to FADD DD is essential to promote catalysis. Strikingly, this can be achieved by a singlemutation, S286Y SseK1 or N302Y SseK2 , or by a combination of multiple mutations in different regions of the enzyme. Nevertheless, these single mutations are enough to account for the best k cat of all mutants, implying that the Tyr residue in that position might also play a catalytic role. Molecular dynamics simulations determine that the mutation S286Y SseK1 couples binding to catalysis To understand the structural role of the single mutation S286Y SseK1 in enhancing binding and enabling glycosylation of the FADD DD , we carried out Gaussian accelerated Molecular Fig. 3 Molecular dynamics simulations of the complexes of S286Y SseK1 and NleB1 EPEC with FADD DD . Superposition of MD frames (0.7, 0.8, 0.9, and 1.0 ms) of the complexes. (a and c) show the complex of FADD DD with the S286Y SseK1 single mutant. (b and d) show the complex of FADD DD with NleB1 EPEC . In (a and b) both proteins are in cartoon representation (enzyme in yellow, acceptor FADD DD in cyan), and the side chain at the point of mutation (Tyr286 SseK1 or Tyr284 NleB1 ) is in black sticks. (c and d) show expansions of the key residues at the interface of contact involving the catalytic domain (FADD DD in cyan cartoon representation; Tyr286 SseK1 or Tyr284 NleB1 in black sticks; key S286Y SseK1 or NleB1 EPEC residues in yellow sticks). 12186 |Chem. Sci.,2021,12,12181–12191 © 2021 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Dynamics simulations (GaMD) on 3D molecular models of the complexes of NleB1 EHEC , SseK1 wt and mutants with UDP- GlcNAc-Mn 2+ and FADD DD (see ESI and Fig. S6†). In the case of S286Y SseK1 , Tyr286 maintains similar favorable contacts as those in the complex of NleB1 EPEC with FADD DD (Fig. 3), where its aromatic side chain is inserted into the groove formed by helices a2 and a3 and the loop connecting them, making close contacts with the backbone of Val121 FADD and the side chain of Ile126 FADD . However, for SseK1 wt and S286I SseK1 the side chain at the point of mutation is either too small (Ser286) or too bulky (Ile286) to be properly allocated in the groove between helices a2 and a3, respectively (Fig. S7†). In the case of S286N SseK1 , the side chain is equally not well allocated in the FADD groove, although a persistent hydrogen bond with Asp123 FADD at helix a2 was observed (Fig. S7†). These GaMD simulations results correlate very well with the kinetics measurements for SseK1 wt and mutants, supporting a key role of the interaction of the side chain at the point of mutation with Ile126 FADD from the FADD DD a2–a3 groove, most likely in the form of a favorable enthalpy contribution (Fig. S8†). The GaMD simulations also allowed us to identify an important correlation between the side chain present at the point of mutation and catalysis, by analyzing the internal dynamics of the acceptor site (Arg117 FADD side chain). In NleB1 EPEC , the salt-bridge interaction between the proposed catalytic base (Glu253 NleB1 ) and the guanidinium of Arg117 FADD is maintained and holds the Arg117 in an orientation appropriate for the nucleophilic attack over the beta face. That interaction is not very conserved in the simulations of the other enzymes, and the role of Glu253 NleB1 (Glu255 SseK1 ) is replaced by the carboxylate of another residue, Asp188 SseK1 , which holds the Arg117 side chain in a rather rigid proper orientation all along the simulation time. This only occurs for the mutants that show glycosylating activity, S286Y SseK1 and S286N SseK1 , whereas that interaction is absent in the cases of S286I SseK1 and SseK1 wt (Fig. S8†) where the Arg117 is more dynamic. This is reected in the root-mean-square-uctuations (RMSF) values of Arg117 in the complexes with NleB1 EPEC , S286Y SseK1 , and S286N SseK1 , which showed the lowest values (below 1 ˚ A; Table S4†). GaMD simulations also show that Phe187 SseK1 , Asp188 SseK1 , and Arg191 SseK1 form a stable network of interactions with Arg117 FADD (acceptor) and the sugar nucleotide (donor, Fig. S9†). Asp188 SseK1 and Arg191 SseK1 constitute the Asp/Arg dyad present in other bacterial GTs effectors. 19 These results explain the need for a GT-bound sugar nucleotide to have efficient FADD DD binding (ordered bi–bi mechanism). The identi- ed network of interactions leads to favorable contacts of the carboxylate side chain of Asp188 SseK1 with the acceptor Fig. 4 Enzyme kinetics of NleB1 EHEC /SseK1 wt /SseK2 wt and mutants on DR3 DD . (a) Glycosylation kinetics of NleB1 EHEC /SseK1 wt /SseK2 wt and mutants against DR3 DD . (b) Plots comparing the K m ,k cat and catalytic efficiency (k cat /K m ) of the different NleB1 EHEC /SseK1 wt /SseK2 wt and mutants. Additional kinetic data are shown in Table S5.†Asterisks indicate that the kinetic parameters for N302Y SseK2 are estimated due to its poorer binding to DR3 DD . © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,12181–12191 | 12187 Edge Article Chemical Science Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Arg117 FADD , and of this with the sugar nucleotide. These interactions are conserved in NleB1-FADD DD complex (Fig. S9†). The GaMD simulations support that a single mutation on Ser286 SseK1 to Tyr leads to a favorable coupling between increased affinity and stability of Arg117 FADD orientation, appropriated towards the nucleophilic attack of the anomeric carbon to render inversion of the conguration. This is achieved by a favorable interaction of the Tyr286 SseK1 residue with Ile126 FADD , leading to a stable salt-bridge of the Arg117 FADD guanidinium polar head with the carboxylate of Asp188 SseK1 . The fact that Glu255 SseK1 is far away from Arg117 FADD and that Asp188 SseK1 takes the role of the leading carboxylate in keeping the guanidinium on a proper orientation over the beta face of the GlcNAc residue of the donor substrate, strongly suggests that Asp188 SseK1 might function as the catalytic base in S286Y SseK1 and S286N SseK1 mutants. In fact, the D186A mutation in NleB1 EPEC (the aligned residue in NleB1) has been also reported to be detrimental for NleB1 activity. 19 The N302Y SseK2 mutant is active against DR3 DD Having established the importance of a Tyr residue in binding/ catalysis at position 284 of CrNleB/NleB1 EPEC/EPEC , 286 of SseK1 and 302 of SseK2, we reasoned that the null activity of the SseK2 wt on a NleB substrate could likely be rescued by replacing Asn302 by Tyr. To achieve that, we rst evaluated the activity of NleB1 EHEC , SseK1 wt and SseK2 wt on DR3 DD ,nding out that while NleB1 EHEC , SseK1 wt were active on DR3 DD , SseK2 wt was not (Fig. 4a). In these experiments, the K m ,k cat and the catalytic Fig. 5 Mutations at S286 in SseK1 and N302 in SseK2 impact Salmonella abundance in RAW264.7 cells. RAW 264.7 cells were infected with the indicated Salmonella complementation strains at a multiplicity of infection of 10. Colony counts were enumerated 24 h later. Asterisks indicate significant differences (p< 0.05) between samples, as determined using one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons tests. 12188 |Chem. Sci.,2021,12,12181–12191 © 2021 The Author(s). Published by the Royal Society of Chemistry Chemical Science Edge Article Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online efficiency for NleB1 EHEC were 2.5-, 2.2- and 5.4-fold better than those of SseK1 wt (Fig. 4b and Table S5†). We then performed enzyme kinetics assays on mutants S286I/N/Y SseK1 and N302I/S/Y SseK2 . As found for SseK2 wt on DR3 DD , S286N SseK1 was also inactive on DR3 DD (Fig. 4a, right panel). The other mutants displayed different degrees of initial velocities. Although the initial velocity for the mutants S286I SseK1 and N302I/S SSeK2 was approximately half that of NleB1 EHEC at 140 mM DR3 DD , these mutants did not reach saturated kinetics, preventing us from determining their kinetic parameters. However, we could obtain kinetic parameters for S286Y SseK1 and N302Y SseK2 (Fig. 4a, b, Table S5†). Again, the mutation to Tyr in both enzymes provided DR3 DD saturation curves. The K m s for NleB1 EHEC and S286Y SseK1 were similar while k cat and catalytic efficiency were 1.75-fold better for NleB1 EHEC than S286Y SseK1 . On the contrary, k cat values were similar for NleB1 EHEC and N302Y SseK2 ,differing more in K m and catalytic efficiency (3.3- and 4.1-fold better constants for Nle- B1 EHEC than those for N302Y SseK2 ; Table S5†). Overall, our data with DR3 DD are slightly more complex than the ones for FADD DD , and in particular suggest that a Tyr residue in positions 284 CrNleB/NleB1 , 286 SseK1 and 302 SseK2 is more benecial for enzyme kinetics than a Ser, and the latter over Ile, being an Asn residue in those positions deleterious for activity. The S286Y SseK1 and N302Y SseK2 mutants promote Salmonella enterica survival in macrophages Finally, we rationalized that if Tyr286 SseK1 and Tyr302 SseK2 strikingly improve the kinetic and the thermodynamic parameters, these mutants might also increase the survival of Salmonella enterica strains in infected macrophages. To test this hypothesis, we introduced in a Salmonella strain lacking all SseK GTs the constructs encoding for the expression of SseK1 wt , SseK2 wt , S286Y SseK1 and N302Y SseK2 . The survival of these mutated strains was evaluated in macrophages showing that S286Y SseK1 and N302Y SseK2 improved the proliferation of this bacterium compared to the strains lacking the expression of all SseK GTs or expressing specically either SseK1 wt or SseK2 wt .In addition, the level of proliferation reached by strains expressing S286Y SseK1 and N302Y SseK2 was highly similar to the wild type bacteria that expresses all three GTs (Fig. 5). This result again demonstrates that SseK GTs containing a Tyr residue in 286 SseK1 , 302 SseK2 , and potentially 289 SseK3 are more robust enzymes that lead to bacterial proliferation in macrophages and in turn, virulence. Conclusions Why Salmonella enterica strains encode three functional NleB orthologs with a narrowed substrate selectivity is puzzling. Salmonella enterica differs from EHEC/EPEC E. coli and C. rodentium both in its life cycle and also in the different location of its GTs in host cells. While EHEC/EPEC E. coli or C. rodentium are extracellular pathogens, Salmonella enterica strains are intracellular pathogens. Furthermore, CrNleB/NleB1 EPEC/EHEC / SseK1 are cytoplasmic proteins, while SseK2/SseK3 are Golgiassociated GTs. 15 This provides another way of regulating the types of protein substrates being glycosylated, which for SseK3 is unique as recently reported. 22 Hence, the SseK narrowed substrate selectivity together with the location-dependent glycosylation of SseK1/2/3 wt might endow Salmonella enterica strains with novel ways of hijacking host signaling pathways. However, the selection pressures, if any, driving the differing substrate selectivities among these enzymes, have yet to be investigated. Here, we have addressed the molecular basis of this narrowed substrate selectivity, which relies on a unique secondshell residue, variable between the SseK GTs and located in the interface of the NleB/SseK-protein substrate complex. The mutation of this second-shell residue, either Ser286 SseK1 or Asn302 SseK2 , to Tyr NleB/NleB1 , leads to mutants with optimal kinetic and thermodynamic parameters. The mutants S286Y SseK1 and N302Y SseK2 become active on particular protein substrates such as FADD DD and DR3 DD , respectively, leading potentially to mutants with a broader substrate selectivity as that of CrNleB/NleB1 EPEC/EHEC . This is also supported by the increase in Salmonella abundance in macrophages by strains expressing these mutants. These mutants promote binding and catalysis, likely because the binding of this second-shell residue surrounding residues is coupled to the stability of the interaction between the acceptor Arg with the catalytic base residue. Therefore, the identity of the second-shell residue nely tunes protein substrate selectivity and, in turn, glycosylation, and might explain whether GT substrate selectivity is narrow or broad. Promiscuous GTs act on multiple protein substrates and are found in all animal kingdoms. 4 Several GTs mechanisms have been discovered by combining X-ray crystallography experiments with other biophysical and biochemical techniques. Initiating GTs such as POFUT1/POGLUT1 (also called Rumi) and POFUT2, require folded EGF and TSR repeats, respectively. These GTs share in common that the EGF and TSR repeats are tethered by direct hydrogen bonds, and require EGF and TSR repeats containing minimal consensus sequences that encompass mostly variable residues located in between two cysteine residues. However, POFUT2, and to a lesser extent POFUT1, apply additional strategies to recognize many different protein substrates, leveraging the water molecules present in the interface of the complexes to recognize different repeats. In addition, POGLUT1 and POFUT2 also interact with the repeats by hydrophobic interactions. 31–33 Other initiating GTs, such as OGT and the large family of GalNAc-T isoenzymes (20 in humans) recognize mostly unfolded extended and compact structures of the peptide acceptor substrates, respectively, by establishing hydrogen bonds. Thus, for example, while for OGT a proline (Pro) residue in 2 (promoting the extended conformation) is required for optimal glycosylation, 34 a Pro-X-Pro motif (promoting the compact conformation) contiguous to the preceding Ser/Thr residue usually favors glycosylation. 35 In addition, GalNAc-Ts contain a exible linker located in between the catalytic and the lectin domains, and a exible loop that provides them with different behaviors due to their dissimilar amino acid sequences. While the exible linker is behind the © 2021 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2021,12,12181–12191 | 12189 Edge Article Chemical Science Open Access Article. Published on 19 August 2021. Downloaded on 3/9/2022 1:48:11 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online