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1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from... Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence. Keywords Peptidoglycan · Hydrolase · E. faecalis · AtlE · R6 Biological context of AtlE are therefore likely to also be involved in substrate binding and may ultimately contribute towards the biologi- E. faecalis is a leading cause of nosocomial infection, caus- cal activity of AtlE. ing life-threatening infections in immunocompromised Here, we report the complete assignment of one of the patients and patients with antibiotic-induced dysbiosis conserved repeats from the C-terminal domain of OG1RF (Arias and Murray 2012; Diekema et al. 2019). The viru- AtlE, and the corresponding structure prediction. These lence of E. faecalis and its resistance to antimicrobials is NMR assignments represent a step forward towards the associated with the presence of a surface rhamnopolysac- identification of the cell wall motif recognised by the R1-R6 charide called the Enterococcal Polysaccharide Antigen domain in AtlE, and of how this domain contributes to E. (EPA; Xu et al. 2000). EPA is made of a rhamnan backbone faecalis virulence. substituted by “decorations” that underpin its biological activity (Smith et al. 2019). Although EPA decorations vary between strains, one gene encoding a putative peptidogly- Methods and experiments can hydrolase (AtlE) is ubiquitous, suggesting an important contribution to EPA activity (Fig. 1 A). AtlE contains a sig- Protein expression and purification nal peptide, a glycosyl hydrolase family 25 (GH25) cata- lytic domain, and a C-terminal domain with six conserved Due to the high sequence similarity of the OG1RF R1-R5 repeats (R1-R6) of unknown function (Fig. 1B). The major sequences (Fig. 1 C), the most divergent repeat (R6, resi- peptidoglycan hydrolase expressed in E. faecalis, AtlA (Qin dues 742–819 of AtlE) was selected for cloning, expres- et al. 1998; Mesnage et al. 2008), also has six C-terminal sion, and purification. The sequence encoding the R6 repeats that are essential for substrate binding and overall domain was PCR amplified using oligonucleotides R6_Fw catalytic activity (Eckert et al. 2006). The R1-R6 domains (ATACCATGGCAGCAATCAGTAATATTGACAACTA) and R6_Rev (ATGGGATCCATTCACTTTTTGTACATA- ACGTTTATTTG) and cloned into pET2818 using NcoI and BamH1 sites to generate pET2818_R6. The plasmid Mike P. Williamson encodes an 86-residue polypeptide with a hexahistidine tag m.williamson@sheffield.ac.uk at the C-terminus. School of Biosciences, University of Sheffield, Firth Court, Western Bank, S10 2TN Sheffield, UK 1 3 248 J. L. Davis et al. Fig. 1 Schematic representation of the variable locus which encodes AtlE, and domain organization of the enzyme. (A) atlE is present in all E. faecalis strains within the EPA variable genetic locus, flanked by two conserved genes encoding a glycosyltransferase ( epaR) and a glycerophos- phodiester phosphodiesterase (gdgp). Five model E. faecalis strains are shown as an example (La Rosa et al. 2015). Genes are coloured accorded to predicted function. (B) AtlE in OG1RF is predicted to contain a signal peptide (SP; residues 1–24), a glycohydrolase group 25 (GH25) domain (residues 167–349), and a predicted binding domain (residues 369–814) consisting of six repeating units (R1-R6). (C) The R1-R6 domains are highly conserved. Positively charged residues are highlighted in red, negative residues are highlighted in blue, and polar residues are highlighted in green E. coli Lemo21(DE3) cells were transformed with the UK). Gel filtration was performed on the concentrated pro - pET2818_R6 construct for protein overexpression and tein using a Superdex 75 26/200 column pre-equilibrated purification. Cells were cultivated at 37 °C, shaking, in in buffer C (40 mM phosphate buffer, pH 6.0). Fractions M9 media, containing 2 g/L of C-glucose and 1 g/L of containing R6 were collected and concentrated as described NH Cl as the only carbon and nitrogen sources, respec- above to a final concentration of 1.2 mM. tively. When an OD of 0.7 was reached, R6 expression was induced by the addition of 1 mM isopropyl β-D-1- thiogalactopyranoside at an incubation temperature of NMR experiments 25 °C. After 12 h, cells were harvested by centrifugation at 6000 ⋅ g for 15 min at 4 °C. Pellets were resuspended in 20 All NMR experiments were recorded at 298 K using a Bruker mL of buffer A (50 mM phosphate, 300 mM NaCl, pH 7.5) Neo 600 MHz NMR spectrometer with a 5 mm TCI cryo- supplemented with a Roche cOmplete™ EDTA free prote- probe running TopSpin version 4.0.5. NMR experiments ase inhibitor tablet. Cells were lysed by sonication and spun were performed in 5-mm NMR tubes containing 1 mM R6, at 30,000 ⋅ g for 30 min at 4 °C. The supernatant was then 1 mM trimethylsilylpropanoic acid (TSP), 2 mM sodium loaded onto a 5 mL HisTrap affinity column and equilibrated azide, and 10% v/v H O, in 40 mM phosphate buffer at in five column volumes of buffer A. The His-tagged R6 was pH 6.0, with a total volume of 550 µL. Two-dimensional 15 1 13 1 eluted using a 150 mL 0–100% gradient of buffer A contain - N- H and C- H HSQC, and an assortment of three- ing 500 mM imidazole and concentrated using a Vivaspin dimensional NMR experiments, HNCO, HNCACO, HNCA, 10,000 MWCO centrifugal concentrator (Generon, Slough, HNCOCA, HNCACB, HNCOCACB, HCCH-TOCSY and 1 3 1 13 15 H, C, and N resonance assignments of a conserved putative cell wall binding domain from Enterococcus… 249 CCH-TOCSY, were performed for the assignment of R6. with clear, well-defined peaks. Excluding the “difficult” The assignment of arginine N -H side-chain resonances signals (N-terminal residue and His-tag, non-protonated required an additional three-dimensional TOCSY-HSQC aliphatic and aromatic C and N, Argη, Lysζ), 98.7% of all experiment, with a mixing time of 120 ms. Using informa- backbone N and amide protons were assigned (missing 15 1 α β ’ α β tion obtained from the assigned N- H HSQC spectrum, only A2), 100% of all C , C , C , H and H backbone sig- HA and HB resonances were assigned from a three-dimen- nals were obtained, as well as 100% of all asparagine side- δ δ1 δ2 ε sional HBHA(CO)NH experiment, which were then used chain N , H and H signals, 100% of all glutamine N , ε1 ε2 ε ε with the HCCH-TOCSY and CCH-TOCSY for sidechain H and H , and 100% of arginine N -H signals. 89.5% assignments. In all cases, standard Bruker pulse sequences of sidechain signals were assigned, with most of the miss- were used. The H chemical shifts were referenced accord- ing signals being from aromatic rings. Arginine side chain ing to the internal H signal of TSP resonating at 0.00 ppm. signals (Fig. 2, green) are folded in the nitrogen dimension. 13 15 C and N chemical shifts were then referenced indirectly The full list of assigned shifts can be found within the Bio- according to nuclei-specific gyromagnetic ratios. MagResBank (http://www.bmrb.wisc.edu) under accession number 51184. The TALOS-N webserver (Shen and Bax 2013) was then Extent of assignment and data deposition used to predict the dynamics and secondary structure of R6 from the reported backbone chemical shifts (Fig. 3 A). 1 15 13 α 2 Chemical shifts corresponding to the H , N, C , The random coil index values (RCI-S ) shown in Fig. 3Ai 13 ß 13 ’ C , C of the R6 backbone were assigned using the stan- indicate R6 may have three dynamic regions, specifically dard triple resonance approach (Gardner and Kay 1998). at the N-terminus, C-terminus and between residues 54 to Spectra were processed and analysed using TopSpin ver- 58. Secondary structure prediction (Fig. 3Aii) suggests R6 sion 4.0.2 and FELIX (FELIX NMR, Inc.). The “asstools” to contain seven β-sheets (β1: K17-V22, β2: R25-Y27, β3: assignment program (Reed et al. 2003) was employed to P38-K40, β4: T44-Y52, β5: R59-T62, β6: G65-T68, β7: align and match spin systems to the R6 sequence for the V74-K76), with a flexible region predicted to fall between 1 α 1 ß assignment of the R6 backbone. H , H and arginine side β4 and β5. ε ε chain resonances (N -H ) were assigned manually follow- Alphafold-2 (AF2) is another protein structure predictor ing the method of Ohlenschläger et al. (1996), using the R6 which allows the accurate prediction of protein structures backbone assignments as reference. Sidechain resonances using only the primary sequence (Jumper et al. 2021). In were assigned using HCCH-TOCSY and CCH-TOCSY theory this platform could therefore be used in tandem with experiments. TALOS-N as a validation method. AF2 predicted a simi- Figure 2 shows the assigned N HSQC spectrum for the lar overall protein secondary structure for R6, as compared recombinant R6 protein. The spectrum is of high resolution to the TALOS-N output, as shown in Fig. 3B. Whilst eight 15 1 Fig. 2 Assigned N- H HSQC spectrum of the AtlE R6 sequence, at 298 K in 40 mM phosphate buffer (pH 6), 90% H O, 10% D O. Signals are 2 2 labelled according to amino acid one letter code and posi- tion in the primary sequence. Backbone NH signals are in black, asparagine and glutamine side chain NH pairs are in blue, connected by a dashed line, and arginine NHε signals are in green. The NHε signal of R46 is not shown, due to low signal on this plot 1 3 250 J. L. Davis et al. Fig. 3 Protein dynamics and structure prediction of the AtlE R6 sequence. TALOS-N and the reported backbone chemical shifts were used to (i) calculate the random coil index order parameter (RCI-S ), and (ii) predict the secondary structure of each R6 residue. Using TALOS-N, R6 was predicted to contain only β-sheets. (B) AF2 was also employed to predict the secondary structure of R6 (orange), using the primary sequence alone, and compared against the TALOS-N predic- tion (cyan). In the case of AF2, one ⍺-helix (rectangle) and seven β-sheets (arrows) were predicted to make up the R6 secondary structure. (C) Some differences were observed between the TALOS-N and AF2 secondary structure predictions. (i) Specifically, the AF2 struc - ture (orange) predicted a break in the β-sheet between residues 46–48 (circled in black), which is not seen in the TALOS-N prediction (cyan). (ii) Torsion angles predicted by TALOS-N at residues 46, 47 and 48 β-sheets were predicted rather than seven (β1: K17-M20, In summary, our results suggest that we have produced β2: D24-Y27, β3: G36-V39, β4: G42-R46, β5: Q48-S53, a reliable prediction of the dynamics and secondary struc- β6: G56-T62, β7: G65-T68, β8: V74-K76), the vast major- ture of R6, and that AF2 can be used as a tool to comple- ity were in very similar positions to those predicted by ment chemical shift-based protein structure predictions. TALOS-N. Of interest AF2 predicts a break in the fourth The assignments and structural details reported here will be β-sheet predicted by TALOS-N, between residues 46–48. used to explore the binding of this domain to the cell wall, This discrepancy occurs between two anti-parallel β-sheets to begin to understand the biological activity of AtlE, and in the AF2 tertiary structure (Fig. 3Ci) and may be explained ultimately, its potential contribution to E. faecalis virulence. by the dihedral angles predicted by TALOS-N within this Funding JLD is funded by a BBSRC DTP studentship (grant BB/ region (Fig. 3Cii). Whilst residues 46 and 47 have ϕ and ψ M011151/1, studentship N°2283067). angles characteristic of a β-sheet conformation, residue 48 does not. Taking this into account with the AF2 predicted Data Availability The NMR chemical shift assignments have been tertiary structure, this suggests R6 does have a small break deposited at the Biological Magnetic Resonance Data Bank (http:// www.bmrb.wisc.edu) under the BMRB accession number 51184. within the TALOS-N predicted β4. 1 3 1 13 15 H, C, and N resonance assignments of a conserved putative cell wall binding domain from Enterococcus… 251 Jumper J, Evans R, Pritzel A et al (2021) Highly accurate protein struc- Declarations ture prediction with AlphaFold. Nature 596:583–589. https://doi. org/10.1038/s41586-021-03819-2 Conflict of interest The authors declare that they have no conflict of La Rosa SL, Snipen LG, Murray BE, Willems RJ, Gilmore MS, Diep interest. DB, Nes IF, Brede DA (2015) A genomic virulence reference map of Enterococcus faecalis reveals an important contribution of Ethical standards The experiments described here comply with cur- phage03-like elements in nosocomial genetic lineages to pathoge- rent UK laws. nicity in a Caenorhabditis elegans infection model. Infect Immun 83:2156–2167. https://doi.org/10.1128/IAI.02801-14 Open Access This article is licensed under a Creative Commons Mesnage S, Chau F, Dubost L, Arthur M (2008) Role of N-acetylglu- Attribution 4.0 International License, which permits use, sharing, cosaminidase and N-acetylmuramidase activities in Enterococcus adaptation, distribution and reproduction in any medium or format, faecalis peptidoglycan metabolism. J Biol Chem 283:19845– as long as you give appropriate credit to the original author(s) and the 19853. https://doi.org/10.1074/jbc.M802323200 source, provide a link to the Creative Commons licence, and indicate Ohlenschläger O, Ramachandran R, Flemming J, Gührs KH, Schlott if changes were made. The images or other third party material in this B, Brown LR (1996) NMR secondary structure of the plasmino- article are included in the article’s Creative Commons licence, unless gen activator protein staphylokinase. J Biomol NMR 9:273–286. indicated otherwise in a credit line to the material. If material is not https://doi.org/10.1023/a:1018678925512 included in the article’s Creative Commons licence and your intended Reed MA, Hounslow AM, Sze KH, Barsukov IG, Hosszu LL, Clarke use is not permitted by statutory regulation or exceeds the permitted AR, Craven CJ, Waltho JP (2003) Effects of domain dissection use, you will need to obtain permission directly from the copyright on the folding and stability of the 43 kDa protein PGK probed holder. To view a copy of this licence, visit http://creativecommons. by NMR. J Mol Biol 330:1189–1201. https://doi.org/10.1016/ org/licenses/by/4.0/. s0022-2836(03)00625-9 Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR, 56, 227–241(2013). https://doi. References org/10.1007/s10858-013-9741-y Smith RE, Salamaga B, Szkuta P, Hajdamowicz N, Prajsnar TK, Arias CA, Murray BE (2012) The rise of the Enterococcus: beyond Bulmer GS, Fontaine T, Kołodziejczyk J, Herry JM, Hounslow vancomycin resistance. Nat Rev Microbiol 10:266–278. https:// AM, Williamson MP, Serror P, Mesnage S (2019) Decoration doi.org/10.1038/nrmicro2761 of the enterococcal polysaccharide antigen EPA is essential for Diekema DJ, Hsueh PR, Mendes RE, Pfaller MA, Rolston KV, Sader virulence, cell surface charge and interaction with effectors of the HS, Jones RN (2019) The microbiology of bloodstream infection: innate immune system. PLOS Pathog 15:1007730. https://doi. 20-Year trends from the SENTRY antimicrobial surveillance pro- org/10.1371/journal.ppat.1007730 gram. Antimicrob Agents Chemother 63:e00355–e00319. https:// Qin X, Singh KV, Xu Y, Weinstock GM, Murray BE (1998) Effect doi.org/10.1128/AAC.00355-19 of disruption of a gene encoding an autolysin of Enterococcus Eckert C, Lecerf M, Dubost L, Arthur M, Mesnage S (2006) Func- faecalis OG1RF. Antimicrob Agents Chemother 42:2883–2888. tional analysis of AtlA, the major N-acetylglucosaminidase of https://doi.org/10.1128/AAC.42.11.2883 Enterococcus faecalis. J Bacteriol 188:8513–8519. https://doi. Xu Y, Singh KV, Qin X, Murray BE, Weinstock GM (2000) Analy- org/10.1128/JB.01145-06 sis of a gene cluster of Enterococcus faecalis involved in poly- 2 13 15 Gardner KH, Kay LE (1998) The use of H, C, N multidimensional saccharide biosynthesis. Infect Immun 68:815–823. https://doi. NMR to study the structure and dynamics of proteins. Annu Rev org/10.1128/iai.68.2.815-823.2000 Biophys Biomol Struct 27:357–406. https://doi.org/10.1146/ annurev.biophys.27.1.357 Publisher’s note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional affiliations. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis

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Copyright © The Author(s) 2022
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1874-2718
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10.1007/s12104-022-10087-2
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Abstract

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence. Keywords Peptidoglycan · Hydrolase · E. faecalis · AtlE · R6 Biological context of AtlE are therefore likely to also be involved in substrate binding and may ultimately contribute towards the biologi- E. faecalis is a leading cause of nosocomial infection, caus- cal activity of AtlE. ing life-threatening infections in immunocompromised Here, we report the complete assignment of one of the patients and patients with antibiotic-induced dysbiosis conserved repeats from the C-terminal domain of OG1RF (Arias and Murray 2012; Diekema et al. 2019). The viru- AtlE, and the corresponding structure prediction. These lence of E. faecalis and its resistance to antimicrobials is NMR assignments represent a step forward towards the associated with the presence of a surface rhamnopolysac- identification of the cell wall motif recognised by the R1-R6 charide called the Enterococcal Polysaccharide Antigen domain in AtlE, and of how this domain contributes to E. (EPA; Xu et al. 2000). EPA is made of a rhamnan backbone faecalis virulence. substituted by “decorations” that underpin its biological activity (Smith et al. 2019). Although EPA decorations vary between strains, one gene encoding a putative peptidogly- Methods and experiments can hydrolase (AtlE) is ubiquitous, suggesting an important contribution to EPA activity (Fig. 1 A). AtlE contains a sig- Protein expression and purification nal peptide, a glycosyl hydrolase family 25 (GH25) cata- lytic domain, and a C-terminal domain with six conserved Due to the high sequence similarity of the OG1RF R1-R5 repeats (R1-R6) of unknown function (Fig. 1B). The major sequences (Fig. 1 C), the most divergent repeat (R6, resi- peptidoglycan hydrolase expressed in E. faecalis, AtlA (Qin dues 742–819 of AtlE) was selected for cloning, expres- et al. 1998; Mesnage et al. 2008), also has six C-terminal sion, and purification. The sequence encoding the R6 repeats that are essential for substrate binding and overall domain was PCR amplified using oligonucleotides R6_Fw catalytic activity (Eckert et al. 2006). The R1-R6 domains (ATACCATGGCAGCAATCAGTAATATTGACAACTA) and R6_Rev (ATGGGATCCATTCACTTTTTGTACATA- ACGTTTATTTG) and cloned into pET2818 using NcoI and BamH1 sites to generate pET2818_R6. The plasmid Mike P. Williamson encodes an 86-residue polypeptide with a hexahistidine tag m.williamson@sheffield.ac.uk at the C-terminus. School of Biosciences, University of Sheffield, Firth Court, Western Bank, S10 2TN Sheffield, UK 1 3 248 J. L. Davis et al. Fig. 1 Schematic representation of the variable locus which encodes AtlE, and domain organization of the enzyme. (A) atlE is present in all E. faecalis strains within the EPA variable genetic locus, flanked by two conserved genes encoding a glycosyltransferase ( epaR) and a glycerophos- phodiester phosphodiesterase (gdgp). Five model E. faecalis strains are shown as an example (La Rosa et al. 2015). Genes are coloured accorded to predicted function. (B) AtlE in OG1RF is predicted to contain a signal peptide (SP; residues 1–24), a glycohydrolase group 25 (GH25) domain (residues 167–349), and a predicted binding domain (residues 369–814) consisting of six repeating units (R1-R6). (C) The R1-R6 domains are highly conserved. Positively charged residues are highlighted in red, negative residues are highlighted in blue, and polar residues are highlighted in green E. coli Lemo21(DE3) cells were transformed with the UK). Gel filtration was performed on the concentrated pro - pET2818_R6 construct for protein overexpression and tein using a Superdex 75 26/200 column pre-equilibrated purification. Cells were cultivated at 37 °C, shaking, in in buffer C (40 mM phosphate buffer, pH 6.0). Fractions M9 media, containing 2 g/L of C-glucose and 1 g/L of containing R6 were collected and concentrated as described NH Cl as the only carbon and nitrogen sources, respec- above to a final concentration of 1.2 mM. tively. When an OD of 0.7 was reached, R6 expression was induced by the addition of 1 mM isopropyl β-D-1- thiogalactopyranoside at an incubation temperature of NMR experiments 25 °C. After 12 h, cells were harvested by centrifugation at 6000 ⋅ g for 15 min at 4 °C. Pellets were resuspended in 20 All NMR experiments were recorded at 298 K using a Bruker mL of buffer A (50 mM phosphate, 300 mM NaCl, pH 7.5) Neo 600 MHz NMR spectrometer with a 5 mm TCI cryo- supplemented with a Roche cOmplete™ EDTA free prote- probe running TopSpin version 4.0.5. NMR experiments ase inhibitor tablet. Cells were lysed by sonication and spun were performed in 5-mm NMR tubes containing 1 mM R6, at 30,000 ⋅ g for 30 min at 4 °C. The supernatant was then 1 mM trimethylsilylpropanoic acid (TSP), 2 mM sodium loaded onto a 5 mL HisTrap affinity column and equilibrated azide, and 10% v/v H O, in 40 mM phosphate buffer at in five column volumes of buffer A. The His-tagged R6 was pH 6.0, with a total volume of 550 µL. Two-dimensional 15 1 13 1 eluted using a 150 mL 0–100% gradient of buffer A contain - N- H and C- H HSQC, and an assortment of three- ing 500 mM imidazole and concentrated using a Vivaspin dimensional NMR experiments, HNCO, HNCACO, HNCA, 10,000 MWCO centrifugal concentrator (Generon, Slough, HNCOCA, HNCACB, HNCOCACB, HCCH-TOCSY and 1 3 1 13 15 H, C, and N resonance assignments of a conserved putative cell wall binding domain from Enterococcus… 249 CCH-TOCSY, were performed for the assignment of R6. with clear, well-defined peaks. Excluding the “difficult” The assignment of arginine N -H side-chain resonances signals (N-terminal residue and His-tag, non-protonated required an additional three-dimensional TOCSY-HSQC aliphatic and aromatic C and N, Argη, Lysζ), 98.7% of all experiment, with a mixing time of 120 ms. Using informa- backbone N and amide protons were assigned (missing 15 1 α β ’ α β tion obtained from the assigned N- H HSQC spectrum, only A2), 100% of all C , C , C , H and H backbone sig- HA and HB resonances were assigned from a three-dimen- nals were obtained, as well as 100% of all asparagine side- δ δ1 δ2 ε sional HBHA(CO)NH experiment, which were then used chain N , H and H signals, 100% of all glutamine N , ε1 ε2 ε ε with the HCCH-TOCSY and CCH-TOCSY for sidechain H and H , and 100% of arginine N -H signals. 89.5% assignments. In all cases, standard Bruker pulse sequences of sidechain signals were assigned, with most of the miss- were used. The H chemical shifts were referenced accord- ing signals being from aromatic rings. Arginine side chain ing to the internal H signal of TSP resonating at 0.00 ppm. signals (Fig. 2, green) are folded in the nitrogen dimension. 13 15 C and N chemical shifts were then referenced indirectly The full list of assigned shifts can be found within the Bio- according to nuclei-specific gyromagnetic ratios. MagResBank (http://www.bmrb.wisc.edu) under accession number 51184. The TALOS-N webserver (Shen and Bax 2013) was then Extent of assignment and data deposition used to predict the dynamics and secondary structure of R6 from the reported backbone chemical shifts (Fig. 3 A). 1 15 13 α 2 Chemical shifts corresponding to the H , N, C , The random coil index values (RCI-S ) shown in Fig. 3Ai 13 ß 13 ’ C , C of the R6 backbone were assigned using the stan- indicate R6 may have three dynamic regions, specifically dard triple resonance approach (Gardner and Kay 1998). at the N-terminus, C-terminus and between residues 54 to Spectra were processed and analysed using TopSpin ver- 58. Secondary structure prediction (Fig. 3Aii) suggests R6 sion 4.0.2 and FELIX (FELIX NMR, Inc.). The “asstools” to contain seven β-sheets (β1: K17-V22, β2: R25-Y27, β3: assignment program (Reed et al. 2003) was employed to P38-K40, β4: T44-Y52, β5: R59-T62, β6: G65-T68, β7: align and match spin systems to the R6 sequence for the V74-K76), with a flexible region predicted to fall between 1 α 1 ß assignment of the R6 backbone. H , H and arginine side β4 and β5. ε ε chain resonances (N -H ) were assigned manually follow- Alphafold-2 (AF2) is another protein structure predictor ing the method of Ohlenschläger et al. (1996), using the R6 which allows the accurate prediction of protein structures backbone assignments as reference. Sidechain resonances using only the primary sequence (Jumper et al. 2021). In were assigned using HCCH-TOCSY and CCH-TOCSY theory this platform could therefore be used in tandem with experiments. TALOS-N as a validation method. AF2 predicted a simi- Figure 2 shows the assigned N HSQC spectrum for the lar overall protein secondary structure for R6, as compared recombinant R6 protein. The spectrum is of high resolution to the TALOS-N output, as shown in Fig. 3B. Whilst eight 15 1 Fig. 2 Assigned N- H HSQC spectrum of the AtlE R6 sequence, at 298 K in 40 mM phosphate buffer (pH 6), 90% H O, 10% D O. Signals are 2 2 labelled according to amino acid one letter code and posi- tion in the primary sequence. Backbone NH signals are in black, asparagine and glutamine side chain NH pairs are in blue, connected by a dashed line, and arginine NHε signals are in green. The NHε signal of R46 is not shown, due to low signal on this plot 1 3 250 J. L. Davis et al. Fig. 3 Protein dynamics and structure prediction of the AtlE R6 sequence. TALOS-N and the reported backbone chemical shifts were used to (i) calculate the random coil index order parameter (RCI-S ), and (ii) predict the secondary structure of each R6 residue. Using TALOS-N, R6 was predicted to contain only β-sheets. (B) AF2 was also employed to predict the secondary structure of R6 (orange), using the primary sequence alone, and compared against the TALOS-N predic- tion (cyan). In the case of AF2, one ⍺-helix (rectangle) and seven β-sheets (arrows) were predicted to make up the R6 secondary structure. (C) Some differences were observed between the TALOS-N and AF2 secondary structure predictions. (i) Specifically, the AF2 struc - ture (orange) predicted a break in the β-sheet between residues 46–48 (circled in black), which is not seen in the TALOS-N prediction (cyan). (ii) Torsion angles predicted by TALOS-N at residues 46, 47 and 48 β-sheets were predicted rather than seven (β1: K17-M20, In summary, our results suggest that we have produced β2: D24-Y27, β3: G36-V39, β4: G42-R46, β5: Q48-S53, a reliable prediction of the dynamics and secondary struc- β6: G56-T62, β7: G65-T68, β8: V74-K76), the vast major- ture of R6, and that AF2 can be used as a tool to comple- ity were in very similar positions to those predicted by ment chemical shift-based protein structure predictions. TALOS-N. Of interest AF2 predicts a break in the fourth The assignments and structural details reported here will be β-sheet predicted by TALOS-N, between residues 46–48. used to explore the binding of this domain to the cell wall, This discrepancy occurs between two anti-parallel β-sheets to begin to understand the biological activity of AtlE, and in the AF2 tertiary structure (Fig. 3Ci) and may be explained ultimately, its potential contribution to E. faecalis virulence. by the dihedral angles predicted by TALOS-N within this Funding JLD is funded by a BBSRC DTP studentship (grant BB/ region (Fig. 3Cii). Whilst residues 46 and 47 have ϕ and ψ M011151/1, studentship N°2283067). angles characteristic of a β-sheet conformation, residue 48 does not. Taking this into account with the AF2 predicted Data Availability The NMR chemical shift assignments have been tertiary structure, this suggests R6 does have a small break deposited at the Biological Magnetic Resonance Data Bank (http:// www.bmrb.wisc.edu) under the BMRB accession number 51184. within the TALOS-N predicted β4. 1 3 1 13 15 H, C, and N resonance assignments of a conserved putative cell wall binding domain from Enterococcus… 251 Jumper J, Evans R, Pritzel A et al (2021) Highly accurate protein struc- Declarations ture prediction with AlphaFold. Nature 596:583–589. https://doi. org/10.1038/s41586-021-03819-2 Conflict of interest The authors declare that they have no conflict of La Rosa SL, Snipen LG, Murray BE, Willems RJ, Gilmore MS, Diep interest. DB, Nes IF, Brede DA (2015) A genomic virulence reference map of Enterococcus faecalis reveals an important contribution of Ethical standards The experiments described here comply with cur- phage03-like elements in nosocomial genetic lineages to pathoge- rent UK laws. nicity in a Caenorhabditis elegans infection model. Infect Immun 83:2156–2167. https://doi.org/10.1128/IAI.02801-14 Open Access This article is licensed under a Creative Commons Mesnage S, Chau F, Dubost L, Arthur M (2008) Role of N-acetylglu- Attribution 4.0 International License, which permits use, sharing, cosaminidase and N-acetylmuramidase activities in Enterococcus adaptation, distribution and reproduction in any medium or format, faecalis peptidoglycan metabolism. J Biol Chem 283:19845– as long as you give appropriate credit to the original author(s) and the 19853. https://doi.org/10.1074/jbc.M802323200 source, provide a link to the Creative Commons licence, and indicate Ohlenschläger O, Ramachandran R, Flemming J, Gührs KH, Schlott if changes were made. The images or other third party material in this B, Brown LR (1996) NMR secondary structure of the plasmino- article are included in the article’s Creative Commons licence, unless gen activator protein staphylokinase. J Biomol NMR 9:273–286. indicated otherwise in a credit line to the material. If material is not https://doi.org/10.1023/a:1018678925512 included in the article’s Creative Commons licence and your intended Reed MA, Hounslow AM, Sze KH, Barsukov IG, Hosszu LL, Clarke use is not permitted by statutory regulation or exceeds the permitted AR, Craven CJ, Waltho JP (2003) Effects of domain dissection use, you will need to obtain permission directly from the copyright on the folding and stability of the 43 kDa protein PGK probed holder. To view a copy of this licence, visit http://creativecommons. by NMR. J Mol Biol 330:1189–1201. https://doi.org/10.1016/ org/licenses/by/4.0/. s0022-2836(03)00625-9 Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR, 56, 227–241(2013). https://doi. 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Annu Rev org/10.1128/iai.68.2.815-823.2000 Biophys Biomol Struct 27:357–406. https://doi.org/10.1146/ annurev.biophys.27.1.357 Publisher’s note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional affiliations. 1 3

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Biomolecular NMR AssignmentsSpringer Journals

Published: Oct 1, 2022

Keywords: Peptidoglycan; Hydrolase; E. faecalis; AtlE; R6

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