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Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution

Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal... The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a sug- gested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation dur- 15 13 1 ing biofilm formation. Here we report the near complete N/ C/ H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic N-T, T and NOE 1 2 data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding. Keywords NMR assignments · Pneumococcal serine rich repeat protein · Secondary structure · X-ray comparison · Backbone dynamics Biological context Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1210 4-020-09944 -9) contains supplementary material, which is available to authorized users. The Gram-positive commensal and human-adapted bacte- rium Streptococcus pneumoniae colonizes the upper respira- * Peter Agback tory tract in about 10% of healthy adults and up to 60% of peter.agback@slu.se children, without necessarily causing any symptoms (van Science for Life Laboratory, Department of Medicine, Solna, der Poll and Opal 2009). However, upon certain not yet Karolinska Institute, and Division of Infectious Diseases, well-defined triggers, pneumococcus is transformed from Karolinska University Hospital, SE-171 76 Stockholm, a silent colonizer to a virulent pathogen causing diseases Sweden 2 such as otitis media, sinusitis, pneumonia, septicemia and Division of Protein Engineering, Department of Protein meningitis (Weiser et al. 2018). For efficient colonization Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, AlbaNova University Center, within the nasopharynx, pneumococcus displays a multitude Royal Institute of Technology, and Science for Life of surface-exposed modular adhesive and catalytic proteins Laboratory, SE-100 44 Stockholm, Sweden (Perez-Dorado et al. 2012). Department of Molecular Sciences, Swedish University One such adhesin is the giant pneumococcal serine rich of Agricultural Sciences, PO Box 7015, 750 07 Uppsala, repeat protein (PsrP), that was discovered as a key coloniza- Sweden tion factor present in 60% of pneumococcal strains capable Laboratory of Magnetic Radiospectroscopy, N.N. Vorozhtsov of causing pneumonia in children (Blanchette-Cain et al. Institute of Organic Chemistry, SB RAS, Lavrentiev ave. 9, 2013; Sanchez et  al. 2010). PsrP belongs to a family of Novosibirsk, Russia 630090 Vol.:(0123456789) 1 3 196 T. Schulte et al. serine rich repeat proteins (SRRP) displayed on the surface related to the adhesin CnaA of S. aureus, a microbial surface of Gram-positive bacteria for bacterial attachment to host component recognizing adhesive molecule (MSCRAMM) cells (Lizcano et al. 2012). The C-terminal LPXTG motif of (Deivanayagam et al. 2002; Schulte et al. 2014) (Fig. 1a). PsrP is recognized by Sortases and covalently linked to the MSCRAMMs were defined by a common ligand binding bacterial peptidoglycan cell wall. SRRPs share a long, highly mechanism that is mediated by two adjacent subdomains repetitive and glycosylated C-terminal serine rich-repeat comprising Ig-like folds (Foster et al. 2014). In the struc- (SRR) region that varies in length between 400 and 4000 turally and mechanistically well-described ‘dock, lock and residues. Their functional binding region (BR) domains, latch’ (DLL) binding mode, extracellular matrix-derived which bind to a broad range of targets including extracellular peptide ligands dock to the open apo form of MSCRAMMs DNA (eDNA), glyco-conjugates and keratins, are variable and conformational changes create a closed form, in which in sequence and organized into modular domains (Lizcano the ligands are locked into place (Foster et al. 2014). Most et al. 2012). of the described DLL and associated binding mechanisms The positively charged BR domain of PsrP binds to nega- were derived from X-ray structures of apo- and ligand-bound tively charged helical structures such as keratin-10 (KRT-10) forms of MSCRAMMs, and in-depth biophysical investiga- and eDNA, possibly to promote efficient bacterial attachment tions revealed strong interactions even withstanding forces to the upper respiratory tract and during biofilm formation, in the covalent bond range (Deivanayagam et al. 2002; Xiang respectively (Sanchez et al. 2010; Shivshankar et al. 2009; et  al. 2012; Ross et al. 2012; Milles et al. 2018; Herman Blanchette-Cain et al. 2013; Schulte et al. 2014, 2016). The et al. 2014). crystal structure of the KRT10- and DNA-binding domain While we have presented structural docking models for of PsrP (BR ) revealed a fold topology that is distantly binding of KRT-10 or eDNA to the BR domain of PsrP, 187–385 A A B B ye yes s no no assigned: assigned: 13 13 C C 15 15 N N PP PP PP PP PP PP P P 1 1 H H C C NMR NMR PDB:3ZGH PDB:3ZGH 20 2002 0250 50 300 300 350 350 residue number residue number D D 75 750 0 50 500 0 25 250 0 1 1 0 0 D1...D4 D1...D4 A1 A1 −1 −1 A A C1...C2 C1...C2 −2 −2 F1...F2 F1...F2 B B 0.75 0.75 G G E E 0.50 0.50 loop loop D D 0.25 0.25 helix helix 15 13 1 Fig. 1 Near complete N/ C/ H backbone resonance assignment of coded according to the cartoon representation in panel a. Prior to BR revealed a rigid monomer with secondary structure almost the secondary structure calculations, random coil chemical shifts of 187–385 identical to the X-ray structure (PDB:3ZGH). a The crystal struc- BR , were calculated using POTENCI (results shown in Figure 187–385 ture of BR is visualized as in Fig.  3 of our previous publica- S1) (Nielsen and Mulder 2018; Schwarzinger et al. 2000). d T relax- 187–385 2 15 15 2 tion (Schulte et al. 2014). b Assigned non-proline backbone N and ation times, N-NOEs data as well as the general order parameter S 1 13 HN as well as Cα are shown in green. Non-assigned residues are reveal a rigid barrel with flexible N- and C-termini. Panels b–d are shown in black. Proline residues are highlighted (P). c The second- visualized on a common axis corresponding to the residue numbers ary structure of BR calculated from the NMR chemical shifts of BR. T relaxation times are shown in Figure S2 187–385 187–385 1 was compared to the crystal structure at the residue-level, and color- 1 3 S2 S2 NO NOE E T2 /ms T2 /ms Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal… 197 neither their complex structures nor defined molecular NMR spectroscopy binding mechanisms have been demonstrated. It is also yet unclear whether the previously identified inter-molecular NMR experiments were acquired on Bruker Avance III β-sheet dimer of BR is required for ligand binding. In this spectrometers operating at 14.1 T, equipped with a cryo- 1 13 15 report we present the complete H, C and N backbone enhanced QCI-P probe at a temperature of 298  K. The assignment of the PsrP-BR domain that will allow us to backbone residues were assigned, based on standard 3D identify BR residues that are crucial for ligand binding and TROSY triple resonance experiments. The iterative non- involved in binding-associated conformational dynamics. uniform sampling protocol (NUS) comprised HNCO, Our on-going ligand titration experiments will reveal if HNCA and HN(CO)CA, HN(CA)CO, HN(CO)CACB and BR adopts a binding mode similar to or different from the HNCACB experiments (Jaravine et al. 2008; Orekhov and DLL mode of MSCRAMMs. Jaravine 2011; Salzmann et al. 1998, 1999). A 25% sampling schedule was used for all other 3D spectra, yielding a total acquisition time of 153 h (about 1 week). Targeted acquisi- tion (TA) was used for automatic processing and analysis Methods and experiments of spectra as described previously (Jaravine and Orekhov 2006; Jaravine et al. 2008; Orekhov and Jaravine 2011). This Protein production novel procedure reduces significantly data acquisition and analysis time to assign backbone resonance peaks of proteins A TEV-cleavage site (ENLYFQG) was inserted between (Unnerstale et al. 2016; Agback et al. 2019). The automatic the poly-His tag and the BR domain of the previously assignment was validated manually using CcpNmr Analysis 187−385 described BR construct using sequence and liga- 2.4.2 (Vranken et al. 2005). 187−385 α tion-independent cloning to yield His -TEV-BR H protons were assigned using a 3D HCACO sampling (Li and Elledge 2007; Schulte et al. 2014). The protein schedule comprising 25% NUS and N-hsqc-NOESY, was expressed heterologously in E. coli and purified as C-hsqc-NOESY (Kay et al. 1990, 1992; Schleucher et al. previously described (Schulte et al. 2014). Single N and 1994). Data were processed and assigned by Topspin 4.0.6 15 13 double N/ C labeled proteins were expressed in minimal (Bruker) and CcpNmr Analysis 2.4.2, respectively (Vranken 1 13 15 M9 medium according to published protocols ( NH Cl: et al. 2005). The H, C and N backbone chemical shifts 13 13 15 1 g/L and C d -glucose: 10 g/L) (Sivashanmugam et al. were referred to DSS-d6 directly, while C and N chemi- 2009; Fox and Blommel 2009). Bacteria were grown to cal shifts were referred to indirectly. an OD of about 0.4–0.7 in 4L TB medium, pelleted and Random coil chemical shifts of BR , were calculated 187–385 re-suspended in 400 mL of minimal NMR-growth medium using POTENCI with neighbour correction and subtracted 1 15 13 13 13 α for o/n expression at 25 °C. from the experimental HN, N, Cα, Cβ, C′ and H All purification buffers were based on 20 mM HEPES, chemical shifts (Nielsen and Mulder 2018; Schwarzinger 187−385 300 mM NaCl, 10% glycerol pH 7.5. His -TEV-BR et  al. 2000) (Figure S1). The chemical shift index (CSI) was purified using immobilized metal affinity (IMAC, His- was calculated according to the original method (Wishart 13 13 Trap FF GE-Healthcare) and size exclusion chromatogra- et al. 1992). Residues with consecutive Δδ C′ or Δδ Cα phy (SEC, Superdex 75, GE Healthcare). The poly-His tag values above 0.7 ppm and below − 0.7 ppm indicate alpha was removed by TEV cleavage in HEPES buffer comprising helix, and beta strands, respectively. The opposite is valid 5 mM EDTA and 1 mM DTT. Cleaved protein was passed for Δδ Cβ. The CSI for the three nuclei were averaged and through the Ni-NTA column as flow-through. The final reported as “consensus” CSI. sample was purified over Superdex 75 column in 50 mM T, T and NOEs were determined using sensitivity 1 2 sodium-phosphate buffer pH 5.0, 100 mM NaCl, and con- enhanced TROSY-type pulse-sequences with temperature centrated using centrifugation ultrafiltration. compensation train of pulses after acquisition time (Zhu et al. 2000). T relaxation was determined from the fol- lowing series of relaxation delays: 10, 90, 192, 320, 480, NMR samples preparation 690, 980, 1220 and 1444 ms. T relaxation was measured using CPMG delays of 8.5, 17.0, 25.4, 33.9, 42.4, 50.9, All NMR experiments were performed in the buffer contain- 59.4, 76.3 and 93.3 ms. Both T and T experiments were 1 2 ing 50 mM Na–phosphate pH 5.0, 100 mM NaCl, with added repeated to estimate the experimental fitting to about 2%. 1 mM NaN and 10 (v/v) % D O. The buffer-exchanged pro- The same error was set for the NOE experiment. All spec- 3 2 tein was concentrated to at least 0.7 mM using centrifugation tra were processed by Topspin 4.0.6 (Bruker) and evalu- ultrafiltration and 280 μL was placed in a 5 mm shigemi ated using Dynamics Center 2.1 (Bruker), in which T and tube. T data were fit using mono exponential decay functions. 1 3 198 T. Schulte et al. 78G 42Y 112N A B 5T 103G 193T 2S 182F 149G 66T 87S 126T 71N 29G 105 116 175D 147G 194T 67N 186G 136G * * 65K 134G 185G 141G 51S 30E 22S 178G 99G 70G 113G 130G 199S 184G * 95G 197S 73S 160G 110 3G 174T 38V 88T H 155T * 86T 165F * 166T 135N 192S 96S 54T 118S 4N 50G 28T 72I 161T 31G 121T 74S 195S 37R 145K 81I 90T 138Q 183N * 139M 163T 120N 48N 164S 106N 191S 102S (B) 132T 97D * 68D 153S 146K 117L 125T 128G 36T 100K 56T 98L * 120 109T 159T 173R 148Y 110D 172A * 43K 80S 111K 107Y 156V 89Q * 93T 26V * 170Y 177I 123T 13I 34S 53L 120 18N 40I 129S 92L 179I 60T 152S 19I 94L * * 21K 122S 24T 162D 52K 75M 91M 171A 131Y 188V 150L * 122 79Y H * 142F 196Q 104V 32V 144A * 125 116V 17L 124M 119Y 158I 154W * 187K 168T 62V 7V 127Q 200Q 59V 14N 176R 58T 23E 48N 151T 55F 61Y 76R 105K 33D 189V 63N 140N 44L 180N * 114R 69L 8N 27Y * 25K 20A 130 137A * 181Y 119Y 12A 6I 45K 15A 115Q 198L 10A 57Y 35V 49D 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 8.78.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 ( H) [ppm] 1 15 Fig. 2 H– N TROSY spectrum of BR with assigned residue numbers. Cross peaks labelled with red * belong to multiple conformations 187–385 of N- and C-terminal residues 1 15 NOEs were obtained by dividing the H– N peak intensi- Extent of assignments and data deposition ties in a NOE-enhanced spectrum by the corresponding intensities in an unsaturated spectrum. The order param- Targeted Acquisition (TA) and conventional approaches were eters, S , and the fast internal correlation time, τ were combined to assign 94% of non-proline backbone N and 1 13 13 13 obtained by fitting the relaxation parameters at one field HN, 98% of Cα, 96% of Cβ, 96% of C′ and 91% of non- using the Lipari–Szabo model-free approach with a NH glycine H (Fig. 1b). All assigned chemical shifts are labelled bond length of 1.02 Å and a CSA of − 160 ppm (Hiyama in Fig. 2 and the associated peak table has been deposited to et al. 1988; Lipari and Szabo 1982a, b). BMRB with accession code 50157. The secondary structure In the figures and in the text, the standard nomencla- profile derived from the NMR data was almost identical to ture for amino acids of the carbon atoms was used, where the previously determined crystal structure, thus validating 13 13 Cα is the carbon next to the carbonyl group C′ and our resonance assignment (Fig. 1a, c). In more detail, the pre- 13 13 Cβ is the carbon next to Cα (Markley et al. 1998). The viously determined crystal structure of B R revealed a 187–385 secondary structures obtained from NMR and X-ray crys- fold that is distantly related to the DEv-IgG fold topology of tallography were compared using CSI 3.0 (Hafsa et al. MSCRAMMs (Schulte et al. 2014). The DEv-IgG fold topol- 2015). The crystal structure and NMR data presented in ogy can be described as a compressed barrel composed of two Fig. 1 were visualized using PyMol and the R tidyverse, opposing β-sheets that are formed by β-strands ABED (sheet respectively (Schrödinger 2010; Wickham 2016, 2017). I) and CFG (sheet II), and is distinguished from the IgG-fold 1 3 ( N) [ppm] Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal… 199 otherwise in a credit line to the material. If material is not included in by the insertion of two extra strands between strands D and E the article’s Creative Commons licence and your intended use is not (Deivanayagam et al. 2002; Vengadesan and Narayana 2011). permitted by statutory regulation or exceeds the permitted use, you will In BR , the CFG sheet is heavily distorted by loops and 187–385 need to obtain permission directly from the copyright holder. To view a β-turns, and four strands (D1 to D4) are inserted between copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. strands D and E (Fig. 1a). The NMR assignment revealed that residues T353-A356 between strands F1 and F2 adopted a β-strand in solution, thus combining the two short strands into References a single F strand comprising nine residues (Fig. 1a, c). Subtle Agback P et al (2019) Structural characterization and biological func- differences between the crystal and NMR secondary structures tion of bivalent binding of CD2AP to intrinsically disordered were noticed for turn and disordered regions following strand domain of chikungunya virus nsP3 protein. Virology 537:130– F. The two short strands C1 and C2 were each extended by two 142. https ://doi.org/10.1016/j.virol .2019.08.022 residues in solution, but a short sequence comprising residues Blanchette-Cain K et al (2013) Streptococcus pneumoniae biofilm formation is strain dependent, multifactorial, and associated Y267-G270 in between the two strands was not assigned. Fur- with reduced invasiveness and immunoreactivity during colo- thermore, the short G-strand was identified as unstructured in nization. mBio. https ://doi.org/10.1128/mBio.00745 -13 solution. The N- and C-termini comprising G186-E208 and Deivanayagam CCS, Wann ER, Chen W, Carson M, Rajashankar KR, T359-Q385 respectively, were identified as unstructured. Hook M, Narayana SVL (2002) A novel variant of the immu- noglobulin fold in surface adhesins of Staphylococcus aureus: Dynamic backbone motions of BR on a pico- to nano- 187–385 crystal structure of the fibrinogen-binding MSCRAMM, clump- second timescale were evaluated by determining the longitu- ing factor A. EMBO J 21:6660–6672. https ://doi.org/10.1093/ 15 15 dinal (N T ) and transverse (N T ) relaxation times as well 1 2 emboj /cdf61 9 as steady-state heteronuclear nuclear Overhauser enhancement Foster TJ, Geoghegan JA, Ganesh VK, Hoeoek M (2014) Adhesion, 15 1 15 invasion and evasion: the many functions of the surface proteins ( N NOE) of each H– N amide bond (Figs. 1 and S2). High 15 of Staphylococcus aureus. Nat Rev Microbiol 12:49–62. https T relaxation times, large negative N-NOE values and low ://doi.org/10.1038/nrmic ro316 1 general order parameter S values indicated highly dynamic Fox BG, Blommel PG (2009) Autoinduction of protein expression N- and C-termini of BR (Fig. 1d). Indeed, these regions current protocols in protein science CHAPTER 5:Unit-5.23. 187–385 https ://doi.org/10.1002/04711 40864 .ps052 3s56 were not observed in the previously determined crystal struc- 15 Hafsa NE, Arndt D, Wishart DS (2015) CSI 3.0: a web server for ture. However, low T relaxation times, positive N-NOE identifying secondary and super-secondary structure in proteins values as well as S -values between 0.8 and 1 revealed a rigid using NMR chemical shifts. Nucleic Acids Res 43:W370– structure of the compressed BR barrel (Fig. 1). Further- W377. https ://doi.org/10.1093/nar/gkv49 4 187–385 Herman P, El-Kirat-Chatel S, Beaussart A, Geoghegan JA, Foster more, the correlation time for molecular reorientation (τ ) was TJ, Dufrêne YF (2014) The binding force of the staphylococcal estimated to 13 ns as expected for a 20 kDa protein, indicating adhesin SdrG is remarkably strong. Mol Microbiol 93:356–368. that in solution BR is a monomer. 187–385 https ://doi.org/10.1111/mmi.12663 15 13 1 In conclusion, the near complete N/ C/ H backbone reso- Hiyama Y, Niu CH, Silverton JV, Bavoso A, Torchia DA (1988) Determination of N-15 chemical-shift tensor via N-15-H-2 nance assignment of BR revealed a secondary structure 187–385 dipolar coupling in boc-glycylglycyl[N-15]glycine benzyl ester. prol fi e almost identical to the X-ray structure. BR was 187–385 J Am Chem Soc 110:2378–2383. https://doi.or g/10.1021/ja002 monomeric and rigid in solution exhibiting disordered flexible 16a00 6 N- and C-termini. 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Open Biol 4:130090. https :// doi.org/10.1098/rsob.13009 0 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution

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Abstract

The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a sug- gested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation dur- 15 13 1 ing biofilm formation. Here we report the near complete N/ C/ H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic N-T, T and NOE 1 2 data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding. Keywords NMR assignments · Pneumococcal serine rich repeat protein · Secondary structure · X-ray comparison · Backbone dynamics Biological context Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1210 4-020-09944 -9) contains supplementary material, which is available to authorized users. The Gram-positive commensal and human-adapted bacte- rium Streptococcus pneumoniae colonizes the upper respira- * Peter Agback tory tract in about 10% of healthy adults and up to 60% of peter.agback@slu.se children, without necessarily causing any symptoms (van Science for Life Laboratory, Department of Medicine, Solna, der Poll and Opal 2009). However, upon certain not yet Karolinska Institute, and Division of Infectious Diseases, well-defined triggers, pneumococcus is transformed from Karolinska University Hospital, SE-171 76 Stockholm, a silent colonizer to a virulent pathogen causing diseases Sweden 2 such as otitis media, sinusitis, pneumonia, septicemia and Division of Protein Engineering, Department of Protein meningitis (Weiser et al. 2018). For efficient colonization Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, AlbaNova University Center, within the nasopharynx, pneumococcus displays a multitude Royal Institute of Technology, and Science for Life of surface-exposed modular adhesive and catalytic proteins Laboratory, SE-100 44 Stockholm, Sweden (Perez-Dorado et al. 2012). Department of Molecular Sciences, Swedish University One such adhesin is the giant pneumococcal serine rich of Agricultural Sciences, PO Box 7015, 750 07 Uppsala, repeat protein (PsrP), that was discovered as a key coloniza- Sweden tion factor present in 60% of pneumococcal strains capable Laboratory of Magnetic Radiospectroscopy, N.N. Vorozhtsov of causing pneumonia in children (Blanchette-Cain et al. Institute of Organic Chemistry, SB RAS, Lavrentiev ave. 9, 2013; Sanchez et  al. 2010). PsrP belongs to a family of Novosibirsk, Russia 630090 Vol.:(0123456789) 1 3 196 T. Schulte et al. serine rich repeat proteins (SRRP) displayed on the surface related to the adhesin CnaA of S. aureus, a microbial surface of Gram-positive bacteria for bacterial attachment to host component recognizing adhesive molecule (MSCRAMM) cells (Lizcano et al. 2012). The C-terminal LPXTG motif of (Deivanayagam et al. 2002; Schulte et al. 2014) (Fig. 1a). PsrP is recognized by Sortases and covalently linked to the MSCRAMMs were defined by a common ligand binding bacterial peptidoglycan cell wall. SRRPs share a long, highly mechanism that is mediated by two adjacent subdomains repetitive and glycosylated C-terminal serine rich-repeat comprising Ig-like folds (Foster et al. 2014). In the struc- (SRR) region that varies in length between 400 and 4000 turally and mechanistically well-described ‘dock, lock and residues. Their functional binding region (BR) domains, latch’ (DLL) binding mode, extracellular matrix-derived which bind to a broad range of targets including extracellular peptide ligands dock to the open apo form of MSCRAMMs DNA (eDNA), glyco-conjugates and keratins, are variable and conformational changes create a closed form, in which in sequence and organized into modular domains (Lizcano the ligands are locked into place (Foster et al. 2014). Most et al. 2012). of the described DLL and associated binding mechanisms The positively charged BR domain of PsrP binds to nega- were derived from X-ray structures of apo- and ligand-bound tively charged helical structures such as keratin-10 (KRT-10) forms of MSCRAMMs, and in-depth biophysical investiga- and eDNA, possibly to promote efficient bacterial attachment tions revealed strong interactions even withstanding forces to the upper respiratory tract and during biofilm formation, in the covalent bond range (Deivanayagam et al. 2002; Xiang respectively (Sanchez et al. 2010; Shivshankar et al. 2009; et  al. 2012; Ross et al. 2012; Milles et al. 2018; Herman Blanchette-Cain et al. 2013; Schulte et al. 2014, 2016). The et al. 2014). crystal structure of the KRT10- and DNA-binding domain While we have presented structural docking models for of PsrP (BR ) revealed a fold topology that is distantly binding of KRT-10 or eDNA to the BR domain of PsrP, 187–385 A A B B ye yes s no no assigned: assigned: 13 13 C C 15 15 N N PP PP PP PP PP PP P P 1 1 H H C C NMR NMR PDB:3ZGH PDB:3ZGH 20 2002 0250 50 300 300 350 350 residue number residue number D D 75 750 0 50 500 0 25 250 0 1 1 0 0 D1...D4 D1...D4 A1 A1 −1 −1 A A C1...C2 C1...C2 −2 −2 F1...F2 F1...F2 B B 0.75 0.75 G G E E 0.50 0.50 loop loop D D 0.25 0.25 helix helix 15 13 1 Fig. 1 Near complete N/ C/ H backbone resonance assignment of coded according to the cartoon representation in panel a. Prior to BR revealed a rigid monomer with secondary structure almost the secondary structure calculations, random coil chemical shifts of 187–385 identical to the X-ray structure (PDB:3ZGH). a The crystal struc- BR , were calculated using POTENCI (results shown in Figure 187–385 ture of BR is visualized as in Fig.  3 of our previous publica- S1) (Nielsen and Mulder 2018; Schwarzinger et al. 2000). d T relax- 187–385 2 15 15 2 tion (Schulte et al. 2014). b Assigned non-proline backbone N and ation times, N-NOEs data as well as the general order parameter S 1 13 HN as well as Cα are shown in green. Non-assigned residues are reveal a rigid barrel with flexible N- and C-termini. Panels b–d are shown in black. Proline residues are highlighted (P). c The second- visualized on a common axis corresponding to the residue numbers ary structure of BR calculated from the NMR chemical shifts of BR. T relaxation times are shown in Figure S2 187–385 187–385 1 was compared to the crystal structure at the residue-level, and color- 1 3 S2 S2 NO NOE E T2 /ms T2 /ms Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal… 197 neither their complex structures nor defined molecular NMR spectroscopy binding mechanisms have been demonstrated. It is also yet unclear whether the previously identified inter-molecular NMR experiments were acquired on Bruker Avance III β-sheet dimer of BR is required for ligand binding. In this spectrometers operating at 14.1 T, equipped with a cryo- 1 13 15 report we present the complete H, C and N backbone enhanced QCI-P probe at a temperature of 298  K. The assignment of the PsrP-BR domain that will allow us to backbone residues were assigned, based on standard 3D identify BR residues that are crucial for ligand binding and TROSY triple resonance experiments. The iterative non- involved in binding-associated conformational dynamics. uniform sampling protocol (NUS) comprised HNCO, Our on-going ligand titration experiments will reveal if HNCA and HN(CO)CA, HN(CA)CO, HN(CO)CACB and BR adopts a binding mode similar to or different from the HNCACB experiments (Jaravine et al. 2008; Orekhov and DLL mode of MSCRAMMs. Jaravine 2011; Salzmann et al. 1998, 1999). A 25% sampling schedule was used for all other 3D spectra, yielding a total acquisition time of 153 h (about 1 week). Targeted acquisi- tion (TA) was used for automatic processing and analysis Methods and experiments of spectra as described previously (Jaravine and Orekhov 2006; Jaravine et al. 2008; Orekhov and Jaravine 2011). This Protein production novel procedure reduces significantly data acquisition and analysis time to assign backbone resonance peaks of proteins A TEV-cleavage site (ENLYFQG) was inserted between (Unnerstale et al. 2016; Agback et al. 2019). The automatic the poly-His tag and the BR domain of the previously assignment was validated manually using CcpNmr Analysis 187−385 described BR construct using sequence and liga- 2.4.2 (Vranken et al. 2005). 187−385 α tion-independent cloning to yield His -TEV-BR H protons were assigned using a 3D HCACO sampling (Li and Elledge 2007; Schulte et al. 2014). The protein schedule comprising 25% NUS and N-hsqc-NOESY, was expressed heterologously in E. coli and purified as C-hsqc-NOESY (Kay et al. 1990, 1992; Schleucher et al. previously described (Schulte et al. 2014). Single N and 1994). Data were processed and assigned by Topspin 4.0.6 15 13 double N/ C labeled proteins were expressed in minimal (Bruker) and CcpNmr Analysis 2.4.2, respectively (Vranken 1 13 15 M9 medium according to published protocols ( NH Cl: et al. 2005). The H, C and N backbone chemical shifts 13 13 15 1 g/L and C d -glucose: 10 g/L) (Sivashanmugam et al. were referred to DSS-d6 directly, while C and N chemi- 2009; Fox and Blommel 2009). Bacteria were grown to cal shifts were referred to indirectly. an OD of about 0.4–0.7 in 4L TB medium, pelleted and Random coil chemical shifts of BR , were calculated 187–385 re-suspended in 400 mL of minimal NMR-growth medium using POTENCI with neighbour correction and subtracted 1 15 13 13 13 α for o/n expression at 25 °C. from the experimental HN, N, Cα, Cβ, C′ and H All purification buffers were based on 20 mM HEPES, chemical shifts (Nielsen and Mulder 2018; Schwarzinger 187−385 300 mM NaCl, 10% glycerol pH 7.5. His -TEV-BR et  al. 2000) (Figure S1). The chemical shift index (CSI) was purified using immobilized metal affinity (IMAC, His- was calculated according to the original method (Wishart 13 13 Trap FF GE-Healthcare) and size exclusion chromatogra- et al. 1992). Residues with consecutive Δδ C′ or Δδ Cα phy (SEC, Superdex 75, GE Healthcare). The poly-His tag values above 0.7 ppm and below − 0.7 ppm indicate alpha was removed by TEV cleavage in HEPES buffer comprising helix, and beta strands, respectively. The opposite is valid 5 mM EDTA and 1 mM DTT. Cleaved protein was passed for Δδ Cβ. The CSI for the three nuclei were averaged and through the Ni-NTA column as flow-through. The final reported as “consensus” CSI. sample was purified over Superdex 75 column in 50 mM T, T and NOEs were determined using sensitivity 1 2 sodium-phosphate buffer pH 5.0, 100 mM NaCl, and con- enhanced TROSY-type pulse-sequences with temperature centrated using centrifugation ultrafiltration. compensation train of pulses after acquisition time (Zhu et al. 2000). T relaxation was determined from the fol- lowing series of relaxation delays: 10, 90, 192, 320, 480, NMR samples preparation 690, 980, 1220 and 1444 ms. T relaxation was measured using CPMG delays of 8.5, 17.0, 25.4, 33.9, 42.4, 50.9, All NMR experiments were performed in the buffer contain- 59.4, 76.3 and 93.3 ms. Both T and T experiments were 1 2 ing 50 mM Na–phosphate pH 5.0, 100 mM NaCl, with added repeated to estimate the experimental fitting to about 2%. 1 mM NaN and 10 (v/v) % D O. The buffer-exchanged pro- The same error was set for the NOE experiment. All spec- 3 2 tein was concentrated to at least 0.7 mM using centrifugation tra were processed by Topspin 4.0.6 (Bruker) and evalu- ultrafiltration and 280 μL was placed in a 5 mm shigemi ated using Dynamics Center 2.1 (Bruker), in which T and tube. T data were fit using mono exponential decay functions. 1 3 198 T. Schulte et al. 78G 42Y 112N A B 5T 103G 193T 2S 182F 149G 66T 87S 126T 71N 29G 105 116 175D 147G 194T 67N 186G 136G * * 65K 134G 185G 141G 51S 30E 22S 178G 99G 70G 113G 130G 199S 184G * 95G 197S 73S 160G 110 3G 174T 38V 88T H 155T * 86T 165F * 166T 135N 192S 96S 54T 118S 4N 50G 28T 72I 161T 31G 121T 74S 195S 37R 145K 81I 90T 138Q 183N * 139M 163T 120N 48N 164S 106N 191S 102S (B) 132T 97D * 68D 153S 146K 117L 125T 128G 36T 100K 56T 98L * 120 109T 159T 173R 148Y 110D 172A * 43K 80S 111K 107Y 156V 89Q * 93T 26V * 170Y 177I 123T 13I 34S 53L 120 18N 40I 129S 92L 179I 60T 152S 19I 94L * * 21K 122S 24T 162D 52K 75M 91M 171A 131Y 188V 150L * 122 79Y H * 142F 196Q 104V 32V 144A * 125 116V 17L 124M 119Y 158I 154W * 187K 168T 62V 7V 127Q 200Q 59V 14N 176R 58T 23E 48N 151T 55F 61Y 76R 105K 33D 189V 63N 140N 44L 180N * 114R 69L 8N 27Y * 25K 20A 130 137A * 181Y 119Y 12A 6I 45K 15A 115Q 198L 10A 57Y 35V 49D 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 8.78.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 ( H) [ppm] 1 15 Fig. 2 H– N TROSY spectrum of BR with assigned residue numbers. Cross peaks labelled with red * belong to multiple conformations 187–385 of N- and C-terminal residues 1 15 NOEs were obtained by dividing the H– N peak intensi- Extent of assignments and data deposition ties in a NOE-enhanced spectrum by the corresponding intensities in an unsaturated spectrum. The order param- Targeted Acquisition (TA) and conventional approaches were eters, S , and the fast internal correlation time, τ were combined to assign 94% of non-proline backbone N and 1 13 13 13 obtained by fitting the relaxation parameters at one field HN, 98% of Cα, 96% of Cβ, 96% of C′ and 91% of non- using the Lipari–Szabo model-free approach with a NH glycine H (Fig. 1b). All assigned chemical shifts are labelled bond length of 1.02 Å and a CSA of − 160 ppm (Hiyama in Fig. 2 and the associated peak table has been deposited to et al. 1988; Lipari and Szabo 1982a, b). BMRB with accession code 50157. The secondary structure In the figures and in the text, the standard nomencla- profile derived from the NMR data was almost identical to ture for amino acids of the carbon atoms was used, where the previously determined crystal structure, thus validating 13 13 Cα is the carbon next to the carbonyl group C′ and our resonance assignment (Fig. 1a, c). In more detail, the pre- 13 13 Cβ is the carbon next to Cα (Markley et al. 1998). The viously determined crystal structure of B R revealed a 187–385 secondary structures obtained from NMR and X-ray crys- fold that is distantly related to the DEv-IgG fold topology of tallography were compared using CSI 3.0 (Hafsa et al. MSCRAMMs (Schulte et al. 2014). The DEv-IgG fold topol- 2015). The crystal structure and NMR data presented in ogy can be described as a compressed barrel composed of two Fig. 1 were visualized using PyMol and the R tidyverse, opposing β-sheets that are formed by β-strands ABED (sheet respectively (Schrödinger 2010; Wickham 2016, 2017). I) and CFG (sheet II), and is distinguished from the IgG-fold 1 3 ( N) [ppm] Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal… 199 otherwise in a credit line to the material. If material is not included in by the insertion of two extra strands between strands D and E the article’s Creative Commons licence and your intended use is not (Deivanayagam et al. 2002; Vengadesan and Narayana 2011). permitted by statutory regulation or exceeds the permitted use, you will In BR , the CFG sheet is heavily distorted by loops and 187–385 need to obtain permission directly from the copyright holder. To view a β-turns, and four strands (D1 to D4) are inserted between copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. strands D and E (Fig. 1a). The NMR assignment revealed that residues T353-A356 between strands F1 and F2 adopted a β-strand in solution, thus combining the two short strands into References a single F strand comprising nine residues (Fig. 1a, c). Subtle Agback P et al (2019) Structural characterization and biological func- differences between the crystal and NMR secondary structures tion of bivalent binding of CD2AP to intrinsically disordered were noticed for turn and disordered regions following strand domain of chikungunya virus nsP3 protein. Virology 537:130– F. The two short strands C1 and C2 were each extended by two 142. https ://doi.org/10.1016/j.virol .2019.08.022 residues in solution, but a short sequence comprising residues Blanchette-Cain K et al (2013) Streptococcus pneumoniae biofilm formation is strain dependent, multifactorial, and associated Y267-G270 in between the two strands was not assigned. Fur- with reduced invasiveness and immunoreactivity during colo- thermore, the short G-strand was identified as unstructured in nization. mBio. https ://doi.org/10.1128/mBio.00745 -13 solution. The N- and C-termini comprising G186-E208 and Deivanayagam CCS, Wann ER, Chen W, Carson M, Rajashankar KR, T359-Q385 respectively, were identified as unstructured. Hook M, Narayana SVL (2002) A novel variant of the immu- noglobulin fold in surface adhesins of Staphylococcus aureus: Dynamic backbone motions of BR on a pico- to nano- 187–385 crystal structure of the fibrinogen-binding MSCRAMM, clump- second timescale were evaluated by determining the longitu- ing factor A. EMBO J 21:6660–6672. https ://doi.org/10.1093/ 15 15 dinal (N T ) and transverse (N T ) relaxation times as well 1 2 emboj /cdf61 9 as steady-state heteronuclear nuclear Overhauser enhancement Foster TJ, Geoghegan JA, Ganesh VK, Hoeoek M (2014) Adhesion, 15 1 15 invasion and evasion: the many functions of the surface proteins ( N NOE) of each H– N amide bond (Figs. 1 and S2). High 15 of Staphylococcus aureus. Nat Rev Microbiol 12:49–62. https T relaxation times, large negative N-NOE values and low ://doi.org/10.1038/nrmic ro316 1 general order parameter S values indicated highly dynamic Fox BG, Blommel PG (2009) Autoinduction of protein expression N- and C-termini of BR (Fig. 1d). Indeed, these regions current protocols in protein science CHAPTER 5:Unit-5.23. 187–385 https ://doi.org/10.1002/04711 40864 .ps052 3s56 were not observed in the previously determined crystal struc- 15 Hafsa NE, Arndt D, Wishart DS (2015) CSI 3.0: a web server for ture. However, low T relaxation times, positive N-NOE identifying secondary and super-secondary structure in proteins values as well as S -values between 0.8 and 1 revealed a rigid using NMR chemical shifts. Nucleic Acids Res 43:W370– structure of the compressed BR barrel (Fig. 1). Further- W377. https ://doi.org/10.1093/nar/gkv49 4 187–385 Herman P, El-Kirat-Chatel S, Beaussart A, Geoghegan JA, Foster more, the correlation time for molecular reorientation (τ ) was TJ, Dufrêne YF (2014) The binding force of the staphylococcal estimated to 13 ns as expected for a 20 kDa protein, indicating adhesin SdrG is remarkably strong. Mol Microbiol 93:356–368. that in solution BR is a monomer. 187–385 https ://doi.org/10.1111/mmi.12663 15 13 1 In conclusion, the near complete N/ C/ H backbone reso- Hiyama Y, Niu CH, Silverton JV, Bavoso A, Torchia DA (1988) Determination of N-15 chemical-shift tensor via N-15-H-2 nance assignment of BR revealed a secondary structure 187–385 dipolar coupling in boc-glycylglycyl[N-15]glycine benzyl ester. prol fi e almost identical to the X-ray structure. BR was 187–385 J Am Chem Soc 110:2378–2383. https://doi.or g/10.1021/ja002 monomeric and rigid in solution exhibiting disordered flexible 16a00 6 N- and C-termini. 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