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

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.

UK). Gel filtration was performed on the concentrated protein using a Superdex 75 26/200 column pre-equilibrated in buffer C (40 mM phosphate buffer, pH 6.0). Fractions containing R6 were collected and concentrated as described above to a final concentration of 1.2 mM.

NMR experiments
All NMR experiments were recorded at 298 K using a Bruker Neo 600 MHz NMR spectrometer with a 5 mm TCI cryoprobe running TopSpin version 4.0.5. NMR experiments were performed in 5-mm NMR tubes containing 1 mM R6, 1 mM trimethylsilylpropanoic acid (TSP), 2 mM sodium azide, and 10% v/v 2 H 2 O, in 40 mM phosphate buffer at pH 6.0, with a total volume of 550 µL. Two-dimensional 15 N-1 H and 13 C-1 H HSQC, and an assortment of threedimensional NMR experiments, HNCO, HNCACO, HNCA, HNCOCA, HNCACB, HNCOCACB, HCCH-TOCSY and E. coli Lemo21(DE3) cells were transformed with the pET2818_R6 construct for protein overexpression and purification. Cells were cultivated at 37 °C, shaking, in M9 media, containing 2 g/L of 13 C-glucose and 1 g/L of 15 NH 4 Cl as the only carbon and nitrogen sources, respectively. When an OD 600 of 0.7 was reached, R6 expression was induced by the addition of 1 mM isopropyl β-D-1thiogalactopyranoside at an incubation temperature of 25 °C. After 12 h, cells were harvested by centrifugation at 6000 ⋅ g for 15 min at 4 °C. Pellets were resuspended in 20 mL of buffer A (50 mM phosphate, 300 mM NaCl, pH 7.5) supplemented with a Roche cOmplete™ EDTA free protease inhibitor tablet. Cells were lysed by sonication and spun at 30,000 ⋅ g for 30 min at 4 °C. The supernatant was then loaded onto a 5 mL HisTrap affinity column and equilibrated in five column volumes of buffer A. The His-tagged R6 was eluted using a 150 mL 0-100% gradient of buffer A containing 500 mM imidazole and concentrated using a Vivaspin 10,000 MWCO centrifugal concentrator (Generon, Slough,  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 1 H, 13 C, and 15 N resonance assignments of a conserved putative cell wall binding domain from Enterococcus… 1 3 with clear, well-defined peaks. Excluding the "difficult" signals (N-terminal residue and His-tag, non-protonated aliphatic and aromatic C and N, Argη, Lysζ), 98.7% of all backbone 15 N and amide protons were assigned (missing only A2), 100% of all C α , C β , C ' , H α and H β backbone signals were obtained, as well as 100% of all asparagine sidechain N δ , H δ1 and H δ2 signals, 100% of all glutamine N ε , H ε1 and H ε2 , and 100% of arginine N ε -H ε signals. 89.5% of sidechain signals were assigned, with most of the missing signals being from aromatic rings. Arginine side chain signals (Fig. 2, green) are folded in the nitrogen dimension. The full list of assigned shifts can be found within the Bio-MagResBank (http://www.bmrb.wisc.edu) under accession number 51184.
Alphafold-2 (AF2) is another protein structure predictor which allows the accurate prediction of protein structures using only the primary sequence (Jumper et al. 2021). In theory this platform could therefore be used in tandem with TALOS-N as a validation method. AF2 predicted a similar overall protein secondary structure for R6, as compared to the TALOS-N output, as shown in Fig. 3B. Whilst eight CCH-TOCSY, were performed for the assignment of R6. The assignment of arginine N ε -H side-chain resonances required an additional three-dimensional TOCSY-HSQC experiment, with a mixing time of 120 ms. Using information obtained from the assigned 15 N-1 H HSQC spectrum, HA and HB resonances were assigned from a three-dimensional HBHA(CO)NH experiment, which were then used with the HCCH-TOCSY and CCH-TOCSY for sidechain assignments. In all cases, standard Bruker pulse sequences were used. The 1 H chemical shifts were referenced according to the internal 1 H signal of TSP resonating at 0.00 ppm. 13 C and 15 N chemical shifts were then referenced indirectly according to nuclei-specific gyromagnetic ratios.

Extent of assignment and data deposition
Chemical shifts corresponding to the 1 H N , 15 N, 13 C α , 13 C ß , 13 C ' of the R6 backbone were assigned using the standard triple resonance approach (Gardner and Kay 1998). Spectra were processed and analysed using TopSpin version 4.0.2 and FELIX (FELIX NMR, Inc.). The "asstools" assignment program (Reed et al. 2003) was employed to align and match spin systems to the R6 sequence for the assignment of the R6 backbone. 1 H α , 1 H ß and arginine side chain resonances (N ε -H ε ) were assigned manually following the method of Ohlenschläger et al. (1996), using the R6 backbone assignments as reference. Sidechain resonances were assigned using HCCH-TOCSY and CCH-TOCSY experiments. Figure 2 shows the assigned 15 N HSQC spectrum for the recombinant R6 protein. The spectrum is of high resolution In summary, our results suggest that we have produced a reliable prediction of the dynamics and secondary structure of R6, and that AF2 can be used as a tool to complement chemical shift-based protein structure predictions. The assignments and structural details reported here will be used to explore the binding of this domain to the cell wall, to begin to understand the biological activity of AtlE, and ultimately, its potential contribution to E. faecalis virulence. β-sheets were predicted rather than seven (β1: K17-M20, β2: D24-Y27, β3: G36-V39, β4: G42-R46, β5: Q48-S53, β6: G56-T62, β7: G65-T68, β8: V74-K76), the vast majority were in very similar positions to those predicted by TALOS-N. Of interest AF2 predicts a break in the fourth β-sheet predicted by TALOS-N, between residues 46-48. This discrepancy occurs between two anti-parallel β-sheets in the AF2 tertiary structure (Fig. 3Ci) and may be explained by the dihedral angles predicted by TALOS-N within this region (Fig. 3Cii). Whilst residues 46 and 47 have ϕ and ψ angles characteristic of a β-sheet conformation, residue 48 does not. Taking this into account with the AF2 predicted tertiary structure, this suggests R6 does have a small break within the TALOS-N predicted β4. 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 2 ), 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 prediction (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 structure (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 1 Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Conflict of interest
The authors declare that they have no conflict of interest.

Ethical standards
The experiments described here comply with current UK laws.
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