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Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called “last-resort” drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. 1 13 15 We report here the near complete H/ C/ N resonance assignment of the catalytic domain of LytM, residues 185–316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall. Keywords Antimicrobial resistance · LytM · Peptidoglycan hydrolase · Staphylococcus aureus Biological context Nigo et al. 2017). To treat (multi)resistant bacterial infec- tions new cures are urgently needed. Staphylococcus aureus is a pathogen of great concern Lysins represent a novel group of potential antibacterial because of its ability to cause life-threatening infections and agents with a new mechanism of action. Lysins are naturally its increasing resistance to antibiotics. Methicillin-resistant occurring bacterial cell wall hydrolyzing enzymes (peptido- S. aureus, MRSA, causes infections hard to treat, but strik- glycan hydrolases, PGHs), which when engaged in thera- ingly, MRSA strains with concomitant resistance to many peutics induce bacteriolysis (Schuch et al. 2022). PGHs are other commonly used groups of antibiotics have emerged. classified according to the specific type of bond they cleave. Most alarmingly, MRSA resistance to vancomycin, line- PG endopeptidases hydrolyze bonds within the peptidic zolid, ceftaroline and daptomycin, the last-resort drugs moieties in the bacterial PG, which in S. aureus consist of approved for the treatment of MRSA, has been reported two stem peptides (Ala-D-iso-Gln-Lys-D-Ala) crosslinked (Hiramatsu 1998; Tsiodras et al. 2001; Mangili et al. 2005; by pentaglycine cross-bridges. The latter is the target of the glycyl-glycine endopeptidase LytM, one of S. aureus auto- lysins (Ramadurai et al. 1999). We have recently assigned the chemical shifts of the Helena Tossavainen and Ilona Pitkänen Equal contribution. LytM N-terminal domain and the linker region, encompass- ing residues 26–184, for the characterization of its struc- Perttu Permi ture and interactions (Pitkänen et al. 2023). LytM catalytic perttu.permi@jyu.fi domain (LytM CAT, residues 185–316), is structurally Department of Biological and Environmental Science, homologous to lysostaphin and other MEROPS M23B fam- University of Jyvaskyla, Jyvaskyla, Finland ily of metallo-endopeptidase catalytic domains (Firczuk et Department of Chemistry, Nanoscience Center, University of al. 2005; Grabowska et al. 2015). These enzymes have in Jyvaskyla, Jyvaskyla, Finland common a characteristic narrow groove formed by a β-sheet Institute of Biotechnology, Helsinki Institute of Life Science, and four surrounding loops. At one end of the groove, a University of Helsinki, Helsinki, Finland 1 3 H. Tossavainen et al. catalytic zinc ion is coordinated by two conserved histidines was eluted and further purified by size exclusion chroma - 2+ and an aspartate. The Zn ion, which polarizes the peptide tography using ÄKTA pure chromatography system (GE bond, and a nucleophilic water molecule activated by two Healthcare) with HiLoad Superdex S75 (16/60) column other conserved histidines act in concert to hydrolyze the (GE Healthcare) in 20 mM sodium phosphate pH 6.5, 50 substrate glycyl-glycine bond (Grabowska et al. 2015). mM NaCl buffer. Protein was concentrated using Amicon Lysostaphin catalytic domain is more active than LytM Ultra-15 centrifugal filter units (Millipore). CAT in S. aureus bacterial lysis (Osipovitch and Griswold 2015). LytM CAT in turn defeats LytU, another S. aureus NMR spectroscopy M23B autolysin (Raulinaitis et al. 2017a, b), in exogenous bacteriolytic activity (Antenucci et al. unpublished data). 0.4 mM LytM catalytic protein preparation, uniformly N, Also, in vitro, the preferred Gly-Gly target bond seems to C labelled in 20 mM sodium phosphate (pH 6.5), with differ between the three enzymes, although comparison is 50 mM NaCl, 0.6 mM ZnCl and 95% H O/5% D O was 2 2 2 not straightforward because of the nature of substrates, sam- used for resonance assignments. Protein backbone reso- ple conditions and techniques (Xu et al. 1997; Odintsov et nances were assigned by analyzing HNCACB, HN(CO) al. 2004; Warfield et al. 2006; Raulinaitis et al. 2017b). Our CACB (Yamazaki et al. 1994), HNCO (Muhandiram and recent study, in which we used identical conditions and tech- Kay 1994), i(HCA)CO(CA)NH (Mäntylahti et al. 2009), niques for lysostaphin and LytM, revealed similarities but HBHA(CO)NH spectra, whereas aliphatic and aromatic also differences in their target bond specificity and substrate side chain assignments were obtained from H(CCO)NH, hydrolysis rates (Antenucci et al. 2023). Indeed, our goal is (H)C(CO)NH, HCCH-COSY, and HB(CBCGCD)HD, 1 15 1 13 to compare and understand how differences in structure and HB(CBCGCDCE)HE, H- N and H- C NOESY spectra dynamics can give rise to functional dissimilarities, which (reviewed in Sattler et al. 1999), respectively. Assignment is essential in the development of PGHs into potent antimi- of methyl-containing residues was accomplished with the crobials. To this end, LytM CAT chemical shift assignments, DE-HCCmHm-TOCSY experiment (Permi et al. 2004). together with those of lysostaphin and LytU (Raulinaitis et The sample was subsequently exchanged into 100% al. 2017a; Tossavainen et al. 2018) allow comparative struc- D O, and the order of disappearance of amide peaks was 1 15 tural, dynamical and interaction studies. followed by measuring H- N HSQC spectra. From this 1 13 sample another set of aliphatic and aromatic region H- C NOESY spectra, as well as 4D HACACON (Tossavainen Methods and experiments et al. 2020) and 4D HACANCOi (Karjalainen et al. 2020) spectra were acquired. Expression and purification of LytM CAT All NMR experiments were performed at 298 K on a Bruker Avance III HD 800 MHz spectrometer equipped 1 13 15 The S. aureus LytM catalytic domain (residues 185–316) with a H, C, N cryogenic TCI probe. NMR data were was cloned into pGEX-2T plasmid and overexpressed in processed using Topspin (Bruker) and analyzed using Escherichia coli strain BL21(DE3) pLysS as a glutathione CcpNmr Analysis v. 2.5.2 (Vranken et al. 2005). S-transferase (GST)-fusion protein with a thrombin cleav- 15 13 age site. To produce uniformly N and C labelled pro- tein, the cells were grown in standard M9 minimal medium Extent of assignments and data deposition supplemented with 100 µg/ml ampicillin, NH Cl (1 g/l) 13 1 15 and C-D-glucose (2 g/l) as the sole nitrogen and carbon LytM CAT H- N HSQC spectrum displays very well sources, respectively. Briefly, overnight bacterial preculture dispersed peaks with a few peaks with noteworthy upfield was expanded to two liters and cells were grown at 37 °C, chemical shifts. Y224 amide proton and side chain ε2 pro- 250 rpm until the OD at 600 nm reached 0.6. Then protein tons of Q244, Q277 have shifts below 5.2 ppm, which is expression was induced by adding 0.5 mM isopropyl β-D- consistent with these interacting with aromatic side chains 1-thiogalactopyranoside (IPTG) and cells were incubated at as seen in the crystal structure of LytM CAT (Firczuk et al. 25 °C, 250 rpm for 16 h. Cells were harvested by centrifuga- 2005). The good dispersion of peaks in LytM CAT spectra tion, resuspended in phosphate-buffered saline (PBS) buffer in general arises from the almost all-beta fold and the large and lysed using EmulsiFlex-C3 high-pressure homogeniser number of aromatic residues in the amino acid sequence (Avestin). Protein was captured using Protino Glutathione (1 Phe, 6 His, 3 Trp, and an enriched amount of tyrosines, Agarose 4B (Macherey-Nagel) according to manufactur- 11). Of note are the side chain N-H peaks of the two zinc- er’s instructions. GST was cleaved in situ using thrombin coordinating histidines, H210 and H293 (Fig. 1c). These are protease (BioPharm Laboratories, LLC). Cleaved protein likely to be visible because metal coordination locks their 1 3 Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM 1 15 Fig. 1 NMR resonance assignments of LytM CAT. a H- N HSQC Red labels indicate side chain peaks. b Enlargements of the crowded spectrum of LytM CAT recorded at 800 MHz H frequency, 298 K. regions indicated with boxes in panel a. c The δ1 side chain peak of 1 15 The upper inset shows the peak of Gln277 side chain Hε21, which has the other zinc-coordinating histidine is visible in a H- N HSQC an unusual upfield chemical shift, 4.33 ppm. The lower inset shows the spectrum in which the N transmitter frequency was set to histidine ε2 side chain peak of H293, one of the zinc-coordinating residues. The side chain protonated nitrogen region, 170 ppm. The intensity of H210 peak is folded, and its true N chemical shift is 167.8 ppm, see panel peak is five times lower than that of H293, which explains why it is not 1 1 15 c. The low-intensity peaks in the middle H region of the spectrum present in the traditional H- N HSQC spectrum. arise from a small amount of unfolded protein present in the sample. tautomeric state, and additionally both are hydrogen-bonded addition to the N-terminal residues 183–185, likely to be to nearby residues, H210 δ1-P200 O and H293 ε2-Q295 unstructured in solution, G206, N238, G242-N243, N251, Oε1, in the crystal structure (Firczuk et al. 2005). The cor- N253, G285-T288, S311 do not show a backbone peak in 1 15 responding peaks were visible also in the HSQC spectra of the H- N HSQC spectrum. Seven of these eleven amides lysostaphin and LytU. are located in the loops surrounding the catalytic groove. However, peak intensities show significant variation, and Notably four consecutive residues in the ten-residue loop sixteen amide peaks have broadened beyond detection. In between strands β7 and β8, which borders the catalytic 1 3 H. Tossavainen et al. Fig. 2 Secondary structure prediction and amide protons protected (PDB ID 2B13, chain A), with blue rectangles indicating helices and 1 15 from exchange. a Secondary structure prediction by TALOS-N, with red arrows strands. b H- N HSQC displaying amide peaks protected blue bars representing helices and red bars strands. On the top is from exchange. The spectrum was acquired ~ eight days after lyophi- depicted the secondary structure present in LytM CAT crystal structure lized LytM CAT had been dissolved in D O. histidines H260 and H291 are not observed. Apart from hydrogen bond to an intramolecular H O molecule, which the N-terminal residues, most of the unassigned side chain in total is stabilized by four hydrogen bonds. In all, LytM resonances are found within this same loop and the catalytic CAT in solution appears to faithfully replicate the structure histidines. The assignment percentages are the following: determined by X-ray crystallography (Firczuk et al. 2005). 1 N 15 H 88% (115 out of 132 non-proline residues), N 91% 13 13 Acknowledgements This work was supported by the Academy of Fin- (125 out of all 137 residues), Cα 96% (131/137), Cβ land and Jane and Aatos Erkko foundation. 97% (114 out of 118 non-glycine residues), and CO 93% (127/137) for backbone resonances and 98% for aliphatic Author contributions IP, LA and CT expressed and purified proteins, 1 15 and 90% for aromatic side chain resonances. The H, N, HT and IP prepared all figures and wrote the initial draft of the manu - script. IP, HT, and PP performed experiments and data analyses. HT C chemical shift assignments for LytM CAT have been and PP conceived of and designed the experiments. All authors read, deposited in the BioMagResBank (http://www.bmrb.wisc. commented and approved the final manuscript. edu) under accession number 52149. Although signal dispersion convincingly suggests a well- Funding This work was supported by the grants from the Academy of folded and stable protein in the current sample conditions, Finland (number 323435) and Jane ja Aatos Erkon Säätiö. Open Access funding provided by University of Jyväskylä (JYU). we further studied its properties by determining its secondary structure based on assigned chemical shifts using TALOS-N Data availability The chemical shift assignments have been deposited (Shen and Bax 2015), and by evaluating hydrogen-to-deu- to the BMRB under the accession code: 52,149. terium (H/D) exchange rates. The secondary structure pre- dicted by chemical shifts well reproduces that observed in Declarations the crystal structure, except for the missing short β strands (G202-Q203, A209-H210, P222-Y224, A306-V307) and Ethics approval and consent to participate Not applicable. the predicted strand for residues R263-V266 (Fig. 2a). In the crystal structure R263 and T265 show strand-like hydro- Consent for publication Not applicable. gen bonding, but T265 ψ angle does not conform to that in Competing interests The authors declare that they have no competing a canonical β strand. conflict of interest. The H/D exchange spectra indicate that LytM CAT has a well-protected core, which resists exchange. After approxi- Open Access This article is licensed under a Creative Commons mately eight days in D O, 28 amide LytM CAT peaks are Attribution 4.0 International License, which permits use, sharing, 1 15 adaptation, distribution and reproduction in any medium or format, still present in the H- N HSQC spectrum (Fig. 2b). Except as long as you give appropriate credit to the original author(s) and the for R296 N-H all these amides are hydrogen bonded, 22 of source, provide a link to the Creative Commons licence, and indicate them in strands and five in residues flanking strands. The if changes were made. The images or other third party material in this persistence of R296 N-H is likely to be explained by its article are included in the article’s Creative Commons licence, unless 1 3 Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM indicated otherwise in a credit line to the material. If material is not (residues 26–184). Biomol NMR Assign. https://doi.org/10.1007/ included in the article’s Creative Commons licence and your intended s12104-023-10151-5 use is not permitted by statutory regulation or exceeds the permitted Ramadurai L, Lockwood KJ, Lockwood J et al (1999) Characteriza- use, you will need to obtain permission directly from the copyright tion of a chromosomally encoded glycylglycine endopeptidase of holder. To view a copy of this licence, visit http://creativecommons. Staphylococcus aureus. Microbiol Read Engl 145(Pt 4):801–808. org/licenses/by/4.0/. https://doi.org/10.1099/13500872-145-4-801 Raulinaitis V, Tossavainen H, Aitio O et al (2017a) 1H, 13 C and 15 N resonance assignments of the new lysostaphin family endopepti- dase catalytic domain from Staphylococcus aureus. Biomol NMR References Assign 11:69–73. https://doi.org/10.1007/s12104-016-9722-7 Raulinaitis V, Tossavainen H, Aitio O et al (2017b) Identification and Antenucci L, Virtanen S, Thapa C, Jartti M, Pitkänen I, Tossavainen structural characterization of LytU, a unique peptidoglycan endo- H, Permi P (2023) Reassessing the substrate specificities of the peptidase from the lysostaphin family. Sci Rep 7:6020. https:// major Staphylococcus aureus peptidoglycan hydrolases lyso- doi.org/10.1038/s41598-017-06135-w staphin and LytM. [Preprint] BioRxiv 2023.10.13.562287. Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multi- https://doi.org/10.1101/2023.10.13.562287 dimensional NMR experiments for the structure determination Firczuk M, Mucha A, Bochtler M (2005) Crystal structures of active of proteins in solution employing pulsed field gradients. Prog LytM. J Mol Biol 354:578–590. https://doi.org/10.1016/j. Nucl Magn Reson Spectrosc 34:93–158. https://doi.org/10.1016/ jmb.2005.09.082 S0079-6565(98)00025-9 Grabowska M, Jagielska E, Czapinska H et al (2015) High resolution Schuch R, Cassino C, Vila-Farres X (2022) Direct Lytic agents: Novel, structure of an M23 peptidase with a substrate analogue. Sci Rep rapidly acting potential Antimicrobial Treatment modalities for 5:14833. https://doi.org/10.1038/srep14833 systemic use in the era of rising Antibiotic Resistance. Front Hiramatsu K (1998) Vancomycin resistance in staphylococci. Drug Microbiol 13. https://doi.org/10.3389/fmicb.2022.841905 Resist Updat Rev Comment Antimicrob Anticancer Chemother Shen Y, Bax A (2015) Protein structural information derived from 1:135–150. https://doi.org/10.1016/s1368-7646(98)80029-0 NMR chemical shift with the neural network program TALOS- Karjalainen M, Tossavainen H, Hellman M, Permi P (2020) HACAN- N. Methods Mol Biol Clifton NJ 1260:17–32. https://doi. COi: a new Hα-detected experiment for backbone resonance org/10.1007/978-1-4939-2239-0_2 assignment of intrinsically disordered proteins. J Biomol NMR Tossavainen H, Raulinaitis V, Kauppinen L et al (2018) Structural and 74:741–752. https://doi.org/10.1007/s10858-020-00347-5 functional insights into lysostaphin-substrate Interaction. Front Mangili A, Bica I, Snydman DR, Hamer DH (2005) Daptomycin-resis- Mol Biosci 5:60. https://doi.org/10.3389/fmolb.2018.00060 tant, methicillin-resistant Staphylococcus aureus bacteremia. Clin Tossavainen H, Salovaara S, Hellman M et al (2020) Dispersion from Infect Dis off Publ Infect Dis Soc Am 40:1058–1060. https://doi. Cα or NH: 4D experiments for backbone resonance assignment org/10.1086/428616 of intrinsically disordered proteins. J Biomol NMR 74:147–159. Mäntylahti S, Tossavainen H, Hellman M, Permi P (2009) An intra- https://doi.org/10.1007/s10858-020-00299-w residual i(HCA)CO(CA)NH experiment for the assignment of Tsiodras S, Gold HS, Sakoulas G et al (2001) Linezolid resistance in main-chain resonances in 15 N, 13 C labeled proteins. J Biomol a clinical isolate of Staphylococcus aureus. Lancet Lond Engl NMR 45:301–310. https://doi.org/10.1007/s10858-009-9373-4 358:207–208. https://doi.org/10.1016/S0140-6736(01)05410-1 Muhandiram DR, Kay LE (1994) Gradient-enhanced triple-resonance Vranken WF, Boucher W, Stevens TJ et al (2005) The CCPN data three-dimensional NMR experiments with improved sensitiv- model for NMR spectroscopy: development of a software pipe- ity. J Magn Reson B 103:203–216. https://doi.org/10.1006/ line. Proteins 59:687–696. https://doi.org/10.1002/prot.20449 jmrb.1994.1032 Warfield R, Bardelang P, Saunders H et al (2006) Internally quenched Nigo M, Diaz L, Carvajal LP et al (2017) Ceftaroline-Resistant, Dap- peptides for the study of lysostaphin: an antimicrobial protease tomycin-Tolerant, and heterogeneous vancomycin-intermediate that kills Staphylococcus aureus. Org Biomol Chem 4:3626– methicillin-resistant Staphylococcus aureus causing infective 3638. https://doi.org/10.1039/b607999g endocarditis. Antimicrob Agents Chemother 61:e01235–e01216. Xu N, Huang Z-H, de Jonge BLM, Gage DA (1997) Structural charac- https://doi.org/10.1128/AAC.01235-16 terization of Peptidoglycan Muropeptides by Matrix-assisted laser Odintsov SG, Sabala I, Marcyjaniak M, Bochtler M (2004) Latent desorption ionization Mass Spectrometry and Postsource Decay LytM at 1.3A resolution. J Mol Biol 335:775–785. https://doi. Analysis. Anal Biochem 248:7–14. https://doi.org/10.1006/ org/10.1016/j.jmb.2003.11.009 abio.1997.2073 Osipovitch DC, Griswold KE (2015) Fusion with a cell wall bind- Yamazaki T, Lee W, Arrowsmith CH et al (1994) A suite of Triple Res- ing domain renders autolysin LytM a potent anti-staphylococ- onance NMR experiments for the Backbone assignment of 15 N, cus aureus agent. FEMS Microbiol Lett 362:1–7. https://doi. 13 C, 2H labeled proteins with high sensitivity. J Am Chem Soc org/10.1093/femsle/fnu035 116:11655–11666. https://doi.org/10.1021/ja00105a005 Permi P, Tossavainen H, Hellman M (2004) Efficient assignment of methyl resonances: enhanced sensitivity by gradient selection Publisher’s Note Springer Nature remains neutral with regard to juris- in a DE-MQ-(H)CCmHm-TOCSY experiment. J Biomol NMR dictional claims in published maps and institutional affiliations. 30:275–282. https://doi.org/10.1007/s10858-004-3222-2 1 13 15 Pitkänen I, Tossavainen H, Permi P (2023) H, C, and N NMR chemical shift assignment of LytM N-terminal domain 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM

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Abstract

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called “last-resort” drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. 1 13 15 We report here the near complete H/ C/ N resonance assignment of the catalytic domain of LytM, residues 185–316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall. Keywords Antimicrobial resistance · LytM · Peptidoglycan hydrolase · Staphylococcus aureus Biological context Nigo et al. 2017). To treat (multi)resistant bacterial infec- tions new cures are urgently needed. Staphylococcus aureus is a pathogen of great concern Lysins represent a novel group of potential antibacterial because of its ability to cause life-threatening infections and agents with a new mechanism of action. Lysins are naturally its increasing resistance to antibiotics. Methicillin-resistant occurring bacterial cell wall hydrolyzing enzymes (peptido- S. aureus, MRSA, causes infections hard to treat, but strik- glycan hydrolases, PGHs), which when engaged in thera- ingly, MRSA strains with concomitant resistance to many peutics induce bacteriolysis (Schuch et al. 2022). PGHs are other commonly used groups of antibiotics have emerged. classified according to the specific type of bond they cleave. Most alarmingly, MRSA resistance to vancomycin, line- PG endopeptidases hydrolyze bonds within the peptidic zolid, ceftaroline and daptomycin, the last-resort drugs moieties in the bacterial PG, which in S. aureus consist of approved for the treatment of MRSA, has been reported two stem peptides (Ala-D-iso-Gln-Lys-D-Ala) crosslinked (Hiramatsu 1998; Tsiodras et al. 2001; Mangili et al. 2005; by pentaglycine cross-bridges. The latter is the target of the glycyl-glycine endopeptidase LytM, one of S. aureus auto- lysins (Ramadurai et al. 1999). We have recently assigned the chemical shifts of the Helena Tossavainen and Ilona Pitkänen Equal contribution. LytM N-terminal domain and the linker region, encompass- ing residues 26–184, for the characterization of its struc- Perttu Permi ture and interactions (Pitkänen et al. 2023). LytM catalytic perttu.permi@jyu.fi domain (LytM CAT, residues 185–316), is structurally Department of Biological and Environmental Science, homologous to lysostaphin and other MEROPS M23B fam- University of Jyvaskyla, Jyvaskyla, Finland ily of metallo-endopeptidase catalytic domains (Firczuk et Department of Chemistry, Nanoscience Center, University of al. 2005; Grabowska et al. 2015). These enzymes have in Jyvaskyla, Jyvaskyla, Finland common a characteristic narrow groove formed by a β-sheet Institute of Biotechnology, Helsinki Institute of Life Science, and four surrounding loops. At one end of the groove, a University of Helsinki, Helsinki, Finland 1 3 H. Tossavainen et al. catalytic zinc ion is coordinated by two conserved histidines was eluted and further purified by size exclusion chroma - 2+ and an aspartate. The Zn ion, which polarizes the peptide tography using ÄKTA pure chromatography system (GE bond, and a nucleophilic water molecule activated by two Healthcare) with HiLoad Superdex S75 (16/60) column other conserved histidines act in concert to hydrolyze the (GE Healthcare) in 20 mM sodium phosphate pH 6.5, 50 substrate glycyl-glycine bond (Grabowska et al. 2015). mM NaCl buffer. Protein was concentrated using Amicon Lysostaphin catalytic domain is more active than LytM Ultra-15 centrifugal filter units (Millipore). CAT in S. aureus bacterial lysis (Osipovitch and Griswold 2015). LytM CAT in turn defeats LytU, another S. aureus NMR spectroscopy M23B autolysin (Raulinaitis et al. 2017a, b), in exogenous bacteriolytic activity (Antenucci et al. unpublished data). 0.4 mM LytM catalytic protein preparation, uniformly N, Also, in vitro, the preferred Gly-Gly target bond seems to C labelled in 20 mM sodium phosphate (pH 6.5), with differ between the three enzymes, although comparison is 50 mM NaCl, 0.6 mM ZnCl and 95% H O/5% D O was 2 2 2 not straightforward because of the nature of substrates, sam- used for resonance assignments. Protein backbone reso- ple conditions and techniques (Xu et al. 1997; Odintsov et nances were assigned by analyzing HNCACB, HN(CO) al. 2004; Warfield et al. 2006; Raulinaitis et al. 2017b). Our CACB (Yamazaki et al. 1994), HNCO (Muhandiram and recent study, in which we used identical conditions and tech- Kay 1994), i(HCA)CO(CA)NH (Mäntylahti et al. 2009), niques for lysostaphin and LytM, revealed similarities but HBHA(CO)NH spectra, whereas aliphatic and aromatic also differences in their target bond specificity and substrate side chain assignments were obtained from H(CCO)NH, hydrolysis rates (Antenucci et al. 2023). Indeed, our goal is (H)C(CO)NH, HCCH-COSY, and HB(CBCGCD)HD, 1 15 1 13 to compare and understand how differences in structure and HB(CBCGCDCE)HE, H- N and H- C NOESY spectra dynamics can give rise to functional dissimilarities, which (reviewed in Sattler et al. 1999), respectively. Assignment is essential in the development of PGHs into potent antimi- of methyl-containing residues was accomplished with the crobials. To this end, LytM CAT chemical shift assignments, DE-HCCmHm-TOCSY experiment (Permi et al. 2004). together with those of lysostaphin and LytU (Raulinaitis et The sample was subsequently exchanged into 100% al. 2017a; Tossavainen et al. 2018) allow comparative struc- D O, and the order of disappearance of amide peaks was 1 15 tural, dynamical and interaction studies. followed by measuring H- N HSQC spectra. From this 1 13 sample another set of aliphatic and aromatic region H- C NOESY spectra, as well as 4D HACACON (Tossavainen Methods and experiments et al. 2020) and 4D HACANCOi (Karjalainen et al. 2020) spectra were acquired. Expression and purification of LytM CAT All NMR experiments were performed at 298 K on a Bruker Avance III HD 800 MHz spectrometer equipped 1 13 15 The S. aureus LytM catalytic domain (residues 185–316) with a H, C, N cryogenic TCI probe. NMR data were was cloned into pGEX-2T plasmid and overexpressed in processed using Topspin (Bruker) and analyzed using Escherichia coli strain BL21(DE3) pLysS as a glutathione CcpNmr Analysis v. 2.5.2 (Vranken et al. 2005). S-transferase (GST)-fusion protein with a thrombin cleav- 15 13 age site. To produce uniformly N and C labelled pro- tein, the cells were grown in standard M9 minimal medium Extent of assignments and data deposition supplemented with 100 µg/ml ampicillin, NH Cl (1 g/l) 13 1 15 and C-D-glucose (2 g/l) as the sole nitrogen and carbon LytM CAT H- N HSQC spectrum displays very well sources, respectively. Briefly, overnight bacterial preculture dispersed peaks with a few peaks with noteworthy upfield was expanded to two liters and cells were grown at 37 °C, chemical shifts. Y224 amide proton and side chain ε2 pro- 250 rpm until the OD at 600 nm reached 0.6. Then protein tons of Q244, Q277 have shifts below 5.2 ppm, which is expression was induced by adding 0.5 mM isopropyl β-D- consistent with these interacting with aromatic side chains 1-thiogalactopyranoside (IPTG) and cells were incubated at as seen in the crystal structure of LytM CAT (Firczuk et al. 25 °C, 250 rpm for 16 h. Cells were harvested by centrifuga- 2005). The good dispersion of peaks in LytM CAT spectra tion, resuspended in phosphate-buffered saline (PBS) buffer in general arises from the almost all-beta fold and the large and lysed using EmulsiFlex-C3 high-pressure homogeniser number of aromatic residues in the amino acid sequence (Avestin). Protein was captured using Protino Glutathione (1 Phe, 6 His, 3 Trp, and an enriched amount of tyrosines, Agarose 4B (Macherey-Nagel) according to manufactur- 11). Of note are the side chain N-H peaks of the two zinc- er’s instructions. GST was cleaved in situ using thrombin coordinating histidines, H210 and H293 (Fig. 1c). These are protease (BioPharm Laboratories, LLC). Cleaved protein likely to be visible because metal coordination locks their 1 3 Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM 1 15 Fig. 1 NMR resonance assignments of LytM CAT. a H- N HSQC Red labels indicate side chain peaks. b Enlargements of the crowded spectrum of LytM CAT recorded at 800 MHz H frequency, 298 K. regions indicated with boxes in panel a. c The δ1 side chain peak of 1 15 The upper inset shows the peak of Gln277 side chain Hε21, which has the other zinc-coordinating histidine is visible in a H- N HSQC an unusual upfield chemical shift, 4.33 ppm. The lower inset shows the spectrum in which the N transmitter frequency was set to histidine ε2 side chain peak of H293, one of the zinc-coordinating residues. The side chain protonated nitrogen region, 170 ppm. The intensity of H210 peak is folded, and its true N chemical shift is 167.8 ppm, see panel peak is five times lower than that of H293, which explains why it is not 1 1 15 c. The low-intensity peaks in the middle H region of the spectrum present in the traditional H- N HSQC spectrum. arise from a small amount of unfolded protein present in the sample. tautomeric state, and additionally both are hydrogen-bonded addition to the N-terminal residues 183–185, likely to be to nearby residues, H210 δ1-P200 O and H293 ε2-Q295 unstructured in solution, G206, N238, G242-N243, N251, Oε1, in the crystal structure (Firczuk et al. 2005). The cor- N253, G285-T288, S311 do not show a backbone peak in 1 15 responding peaks were visible also in the HSQC spectra of the H- N HSQC spectrum. Seven of these eleven amides lysostaphin and LytU. are located in the loops surrounding the catalytic groove. However, peak intensities show significant variation, and Notably four consecutive residues in the ten-residue loop sixteen amide peaks have broadened beyond detection. In between strands β7 and β8, which borders the catalytic 1 3 H. Tossavainen et al. Fig. 2 Secondary structure prediction and amide protons protected (PDB ID 2B13, chain A), with blue rectangles indicating helices and 1 15 from exchange. a Secondary structure prediction by TALOS-N, with red arrows strands. b H- N HSQC displaying amide peaks protected blue bars representing helices and red bars strands. On the top is from exchange. The spectrum was acquired ~ eight days after lyophi- depicted the secondary structure present in LytM CAT crystal structure lized LytM CAT had been dissolved in D O. histidines H260 and H291 are not observed. Apart from hydrogen bond to an intramolecular H O molecule, which the N-terminal residues, most of the unassigned side chain in total is stabilized by four hydrogen bonds. In all, LytM resonances are found within this same loop and the catalytic CAT in solution appears to faithfully replicate the structure histidines. The assignment percentages are the following: determined by X-ray crystallography (Firczuk et al. 2005). 1 N 15 H 88% (115 out of 132 non-proline residues), N 91% 13 13 Acknowledgements This work was supported by the Academy of Fin- (125 out of all 137 residues), Cα 96% (131/137), Cβ land and Jane and Aatos Erkko foundation. 97% (114 out of 118 non-glycine residues), and CO 93% (127/137) for backbone resonances and 98% for aliphatic Author contributions IP, LA and CT expressed and purified proteins, 1 15 and 90% for aromatic side chain resonances. The H, N, HT and IP prepared all figures and wrote the initial draft of the manu - script. IP, HT, and PP performed experiments and data analyses. HT C chemical shift assignments for LytM CAT have been and PP conceived of and designed the experiments. All authors read, deposited in the BioMagResBank (http://www.bmrb.wisc. commented and approved the final manuscript. edu) under accession number 52149. Although signal dispersion convincingly suggests a well- Funding This work was supported by the grants from the Academy of folded and stable protein in the current sample conditions, Finland (number 323435) and Jane ja Aatos Erkon Säätiö. Open Access funding provided by University of Jyväskylä (JYU). we further studied its properties by determining its secondary structure based on assigned chemical shifts using TALOS-N Data availability The chemical shift assignments have been deposited (Shen and Bax 2015), and by evaluating hydrogen-to-deu- to the BMRB under the accession code: 52,149. terium (H/D) exchange rates. The secondary structure pre- dicted by chemical shifts well reproduces that observed in Declarations the crystal structure, except for the missing short β strands (G202-Q203, A209-H210, P222-Y224, A306-V307) and Ethics approval and consent to participate Not applicable. the predicted strand for residues R263-V266 (Fig. 2a). In the crystal structure R263 and T265 show strand-like hydro- Consent for publication Not applicable. gen bonding, but T265 ψ angle does not conform to that in Competing interests The authors declare that they have no competing a canonical β strand. conflict of interest. The H/D exchange spectra indicate that LytM CAT has a well-protected core, which resists exchange. After approxi- Open Access This article is licensed under a Creative Commons mately eight days in D O, 28 amide LytM CAT peaks are Attribution 4.0 International License, which permits use, sharing, 1 15 adaptation, distribution and reproduction in any medium or format, still present in the H- N HSQC spectrum (Fig. 2b). Except as long as you give appropriate credit to the original author(s) and the for R296 N-H all these amides are hydrogen bonded, 22 of source, provide a link to the Creative Commons licence, and indicate them in strands and five in residues flanking strands. The if changes were made. The images or other third party material in this persistence of R296 N-H is likely to be explained by its article are included in the article’s Creative Commons licence, unless 1 3 Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM indicated otherwise in a credit line to the material. If material is not (residues 26–184). Biomol NMR Assign. https://doi.org/10.1007/ included in the article’s Creative Commons licence and your intended s12104-023-10151-5 use is not permitted by statutory regulation or exceeds the permitted Ramadurai L, Lockwood KJ, Lockwood J et al (1999) Characteriza- use, you will need to obtain permission directly from the copyright tion of a chromosomally encoded glycylglycine endopeptidase of holder. To view a copy of this licence, visit http://creativecommons. Staphylococcus aureus. Microbiol Read Engl 145(Pt 4):801–808. org/licenses/by/4.0/. https://doi.org/10.1099/13500872-145-4-801 Raulinaitis V, Tossavainen H, Aitio O et al (2017a) 1H, 13 C and 15 N resonance assignments of the new lysostaphin family endopepti- dase catalytic domain from Staphylococcus aureus. 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J Biomol NMR dictional claims in published maps and institutional affiliations. 30:275–282. https://doi.org/10.1007/s10858-004-3222-2 1 13 15 Pitkänen I, Tossavainen H, Permi P (2023) H, C, and N NMR chemical shift assignment of LytM N-terminal domain 1 3

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Biomolecular NMR AssignmentsSpringer Journals

Published: Nov 2, 2023

Keywords: Antimicrobial resistance; LytM; Peptidoglycan hydrolase; Staphylococcus aureus

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