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NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa

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NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa


The periplasmic space resides between the outer membrane (OM) and cytoplasmic membrane (CM) of Gram-negative bacteria. The OM protects Gram-negative bacteria from environmental hazards such as antibiotics and detergents. At the same time, Gram-negative bacteria require rare essential nutrients such as iron, vitamins that are present in the extracellular environment at very low concentrations. Gram-negative bacteria have evolved active acquisition systems to pass the essential nutrients through OM and CM. Since there is no electrochemical gradient to power the active transport at the OM and no ATP in the periplasmic space, these transporters must extract energy from the CM. These transporters in the OM are termed TonB-dependent transporters (TBDT) because they presumably extract the energy from the proton-motive force (pmf) of the CM via the trans-periplasmic protein, TonB protein (Fig. 1A). The energy transduction is assumed to take place via the TonB complex anchored in CM and consisting of TonB, ExbB, and ExbD proteins. ExbB and ExbD are accessory proteins anchored in the CM that convey the pmf across the CM to TonB (Celia et al., 2016; Clarke, Tari & Vogel, 2001; Krewulak & Vogel, 2008; Postle & Larsen, 2007).

TonB-dependent energy transduction system and structures of differently dissected E. coli TonB.

Figure 1: TonB-dependent energy transduction system and structures of differently dissected E. coli TonB.

(A) A schematic model of the TonB-dependent energy transduction system, which transduces the proton motive force of the CM to TonB-dependent transporters (TBDT) via TonB. (B) Sequence alignment presenting the full-length E. coli TonB sequence and differently dissected CTDs used for the structure determination (labeled with PDB IDs). CTDs are highlighted in gray, and the TM region is indicated by a bar. (C) Ribbon drawings of previously reported E. coli TonB CTD structures. The last β-strands at the C-terminal region with the largest conformational differences among the reported structures are colored in red. 1IHR forms intertwined dimers (Chang et al., 2001) and 1U07 forms dimers connected via the C-terminal extended strand (Ködding et al., 2005) in crystals.

TonB protein mediates the energy transduction from the CM to TBDTs. TonB has an N-terminal transmembrane (TM) domain anchored in the CM (Fig. 1). The TM domain is followed by the central region mostly consisting of Pro-Glu and Pro-Lys repeats (Fig. 1B). Extended conformation of the central region allows the protein to span the periplasmic space between OM and CM (Domingo Köhler et al., 2010). The C-terminal domain (CTD) of TonB protein has a globular structure and interacts with a conserved TonB box motif located at the N-terminus of TBDTs (Cadieux & Kadner, 1999; Pawelek et al., 2006; Peacock et al., 2005; Shultis et al., 2006). Despite a wide variety of models for the energy transduction mechanism by TonB, it is still unclear how TonB protein works in the energy transduction that presumably causes the structural changes of the plug domain in TBDTs to facilitate the transport (Celia et al., 2016; Chimento, Kadner & Wiener, 2005; Gresock et al., 2011; Klebba, 2016; Letain & Postle, 1997; Sverzhinsky et al., 2015; White et al., 2017). The crystal structures of the complexes with two different outer membrane receptors from Escherichia coli BtuB and FhuA suggests direct interactions between β-sheets in the CTD of TonB protein and the TonB box (Shultis et al., 2006; Pawelek et al., 2006). Currently proposed models to induce the structural changes of the plug domain include the mechanical pulling of the plug domain via the interaction with TonB box and CTDs of TonB, which are supported by the atomic force microscopy and molecular dynamics (MD) simulation studies suggesting that the interaction is strong enough to remain stable during the mechanical unfolding of the plug domain (Chimento, Kadner & Wiener, 2005; Gumbart, Wiener & Tajkhorshid, 2007; Hickman et al., 2017). Furthermore, the interaction between a highly conserved positive charge at Arg166 of EcTonB and the negative charge of Glu56 of FhuA receptor is proposed to be involved in the disruption of the plug domain (Pawelek et al., 2006).

Interestingly, several groups have reported distinct conformations of the differently dissected CTD of TonB protein from E. coli solved by X-ray crystallography or NMR spectroscopy (Fig. 1C). The crystal structures of E. coli TonB-CTD consisting of the last 85 or 77 residues were composed of an intertwined dimer conformation with three β-strands and one α-helix (PDB codes: 1IHR and 1QXX, respectively) (Chang et al., 2001; Koedding et al., 2004). Whereas a longer construct consisting of the last 92 residues (EcTonB-92) was monomeric in solution, the crystal structure revealed dimerization via β-strands in the C-terminus (PDB: 1U07) (Ködding et al., 2005). Furthermore, TonB protein from E. coli (EcTonB-137, PDB code: 1XX3) was monomeric as observed in solution NMR experiments (Peacock et al., 2005). CTDs of EcTonB bound to outer membrane receptors BtuB and FhuA take a monomeric conformation (Shultis et al., 2006; Pawelek et al., 2006). Therefore, the biological relevance of the intertwined dimers found in the crystal structures of the shorter fragments of CTDs bearing the last 85 or 77 residues has not been clear (Ködding et al., 2005; Koedding et al., 2004; Postle et al., 2010). In contrast, Electron Spin Resonance (EPR) experiments suggested that a significant population of TonB exists as a dimer in solution while the monomeric form is bound to a TBDT (Freed et al., 2013). The major differences among the monomeric structures of EcTonB are observed around the residues 235–239 (highlighted in red) at the C-terminal region (Fig. 1C). Whereas these residues in the NMR structure of EcTonB-137 fold to an additional β-strand (β6) and forming an anti-parallel sheet with the preceding β5-strand, the same C-terminal end is either extended or largely invisible in the crystal structures (Fig. 1C). Intriguingly, β5-strand (residues 226–231) in EcTonB-CTD directly interacts with TonB box of TBDTs in the crystal structures of EcTonB-CTD/TBDT complexes (Pawelek et al., 2006; Shultis et al., 2006). The β5-strand is not accessible for the proposed interaction in the NMR structures of EcTonB-137 (Peacock et al., 2005) due to the additional β6-strand. Therefore, β6-strand in EcTonB-137 NMR structure has to be exchanged with the strand in TonB box when interacting with TBDTs (Peacock et al., 2005). In contrast, the recent solution NMR structure of the last 92 residues from Helicobacter pyroli TonB (HpTonB-92) showed the disordered C-terminal region and the absence of β6-strand (Ciragan et al., 2016), which is more in line with the crystal structures of EcTonB-CTD reported previously (Ködding et al., 2005; Shultis et al., 2006;Pawelek et al., 2006).

Here, we report the NMR structure and MD simulations of a CTD of TonB protein from Pseudomonas aeruginosa (PaTonB-96) to investigate whether the plasticity of the C-terminal region observed for the reported structures of EcTonB is a common feature of TonBs across different organisms.

Materials and Methods

NMR sample preparation

The C-terminal 96 residues of a TonB protein from P. aeruginosa (UniProt: Q51368) was cloned from genomic DNA (ATCC-47085) as an N-terminal SUMO fusion protein, which was previously termed PsTonB-96 (Guerrero, Ciragan & Iwaï, 2015). The construct expresses a His-tag SUMO fusion protein that produces a 99-residue protein after the SUMO tag removal, which we termed PaTonB-96. PaTonB-96 contains the last 96 residues of PaTonB (247–342) and three residues (SHM) at the N-terminal end from the cloning site. The plasmid (pFGRSF15) coding the gene of PaTonB-96 was transformed into E. coli ER2566 strain (New England Biolabs, Ipswich, MA, USA) for production of doubly 15N, 13C-labeled samples. The transformed E. coli cells were grown overnight at 30 °C in 50 mL LB medium supplemented with 25 μg/mL kanamycin. The cells were spun down at 900 × g for 15 min and gently re-suspended in two L pre-warmed M9 medium supplemented with 25 μg/mL kanamycin, containing 15NH4Cl and 13C6-D-glucose as sole nitrogen and carbon sources, respectively. The cells were grown at 37 °C until an OD600 of 0.6. Then, the temperature was lowered to 30 °C for protein expression. The protein expression was induced with a final concentration of one mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were incubated for additional 5 h before harvesting by centrifugation at 4,900 × g at 4 °C for 15 min. The labeled PaTonB-96 was purified following the previously published protocol (Guerrero, Ciragan & Iwaï, 2015). The purified protein was dialyzed against 20 mM sodium phosphate buffer (pH 6.0). The protein solution of PaTonB-96 was concentrated to one mM for NMR analysis using a centrifugal device. The final sample volume was 250 μL containing 10% D2O.

NMR measurements

NMR measurements for the structure determination were recorded on Varian INOVA 800 MHz equipped with a cryogenically cooled five mm probe head. For the sequential backbone assignment, a standard set of double and triple resonance NMR spectra were recorded at 25 °C, including [1H, 15N]-HSQC, HNCO, HNCA, HNCACB, HN(CO)CA, HN(CA)CO, and CBCA(CO)NH (Sattler, Schleucher & Griesinger, 1999). The aliphatic side-chain assignment was carried out using [1H, 13C]-HSQC, HCCH-COSY, ct-[1H, 13C]-HSQC, HBHA(CO)NH, H(CCCO)NH, (H)CC(CO)NH, 15N-resolved [1H, 1H]-TOCSY, and 15N-edited [1H-1H]-NOESY spectra. The assignments for the aromatic side-chains were based on the spectra of (HB)CB(CGCD)HD, (HB)CB(CGCDCE)HE, aromatic region ct-[1H, 13C]-HSQC and 13C-edited [1H,1H]-NOESY. Data was acquired using VnmrJ (Varian Inc., Palo Alto, CA, USA) and data were processed using the NMRpipe software (Delaglio et al., 1995).

NMR measurements for the backbone dynamics were recorded on a Bruker Avance 850 MHz equipped with a cryogenically cooled probe head. The longitudinal (T1) and transverse (T2) relaxation rates and 15N{1H}-heteronuclear nuclear Overhauser effects (NOEs) for backbone 15N atoms were determined at 25 °C using the well-established NMR experiments (Barbato et al., 1992; Kay, Torchia & Bax, 1989). T1(15N) and T2(15N) relaxation times were determined using the following delay times: 10, 50, 100, 200, 300, 500, 800, 1,000, 1,200, and 2,000 ms for T1 and 16, 64, 96, 128, 156, 196, 224, and 256 ms for CMPG pulse train with one ms interval for T2 relaxation rates, respectively. Relaxation times were obtained by fitting a single exponential decay to peak intensity values: I(t) = I0 × exp (–t/T1) or I0 × exp (−t/T2), where I(t) is the peak volume at a time t. Heteronuclear 15N{1H}-NOEs were obtained with a relaxation delay of 5 s with or without saturation of protons. Heteronuclear 15N{1H}-NOEs (η) were determined from the volumes of the HSQC signals using the ratio of η = I/I0. The relaxation data were processed and analyzed using Bruker Dynamic Center (Version 2.1.8; Bruker Inc., Billerica, MA, USA).

Solution NMR structure determination

The sequence-specific resonance assignment was performed with standard methods using triple resonance NMR experiments and CcpNmr Analysis software (version 2.4.1) (Vranken et al., 2005). The chemical shift values from the sequential resonance assignment were used together with NOE peak lists for the structure calculation with the program CYANA 3.0 (Mumenthaler et al., 1997; Güntert & Buchner, 2015). NOE distance restraints were obtained from 3D 15N- and 13C-edited [1H, 1H]-NOESY spectra with 80-ms mixing time. The conformations of prolines were checked based on CB-CG chemical shift before the structure calculation (Schubert et al., 2002). All prolines were predicted to be in trans conformation except P299 that was set to cis-conformation. The three-dimensional NMR conformers were generated using CYANA 3.0, based on the automated NOESY cross peaks assignment (Güntert, 2004; Güntert, Mumenthaler & Wüthrich, 1997). The restrained energy minimization of the final 20 best conformers was performed using AMBER 14 (Pearlman et al., 1995), and the structures were validated with PSVS 1.5 (Bhattacharya, Tejero & Montelione, 2007). The structural statistics are summarized in Table 1.

Table 1:
Structural statistics of the energy-minimized NMR structure of PaTonB-96.
Completeness of resonance assignments (%)b
Backbone 98.5
Side chain, aliphatic 97.0
Side chain, aromatic 88.0
Distance restraints
Total 1,698
Intraresidue (i = j) 465
Sequential (|i−j| = 1) 483
Medium range (1 < |i−j| < 5) 198
Long range (|i−j| ≥ 5) 552
No. of restraints per residue 17.2
No. of long-range restraints per residue 5.6
Residual restraint violations
Average no. of distance violation per structure
0.1–0.2 Å 1
>0.2 Å 0 (max. 0.14)
Average no. of dihedral angle violations per structure
>2.5° 0
Model qualityc
Rmsd backbone atoms (Å) 1.0
Rmsd heavy atoms (Å) 1.6
Rmsd bond lengths (Å) 0.014
Rmsd bond angles (°) 2.1
MolProbity Ramachandran statisticsc
Most favored regions (%) 97.4
Allowed regions (%) 2.5
Disallowed regions (%) 0.1
Global quality scores (raw/Z score)c
Verify3D 0.34/−1.93
ProsaII 0.41/−0.99
PROCHECK (ϕ–ψ) −0.27/−0.75
PROCHECK (all) −0.23/−1.36
MolProbity clash score 0.18/1.49
Model contents
Ordered residue ranges 247–258, 261–320, 323–338
Total no. of residues 99
BMRB accession number 34235
PDB ID code 6FIP
DOI: 10.7717/peerj.5412/table-1


Structural statistics computed for the ensemble of the 20 deposited NMR conformers.
Calculated from the expected number of resonances, excluding highly exchangeable protons (N-terminal, Lys, amino and Arg guanidino groups, hydroxyls of Ser, Thr, and Tyr), carboxyl groups of Asp and Glu, and nonprotonated aromatic carbons. Backbone atoms: HN, N, Cα, Cβ, Hα, and C.’
Calculated using PSVS version 1.5 (Bhattacharya, Tejero & Montelione, 2007).

MD simulation

The MD simulations were performed with Gromacs5 (Abraham et al., 2015) by using Amber ff99SB-ILDN force field and tip4p water model (Jorgensen et al., 1983; Lindorff-Larsen et al., 2010). The first 10 ns from total 400-ns simulation was considered to be the equilibrium period by monitoring the protein root-mean-square-deviation, inertia tensor eigenvalues, and rotation angles. The rest was used for the analysis since the first 10 ns of simulation trajectories was sufficiently enough to remove the significant fluctuations in these parameters (Ollila, Heikkinen & Iwaï, 2018). The NMR structure determined in this work was used as the initial structure and the secondary structures remained unchanged during the entire simulation period. The temperature was coupled to 25 °C with v-rescale thermostat (Bussi, Donadio & Parrinello, 2007), and the pressure was isotropically set to one bar using Parrinello-Rahman barostat (Parrinello & Rahman, 1981). The time-step was two fs, Lennard-Jones interactions were cut-off at one nm, PME (Darden, York & Pedersen, 1993; Essmann et al., 1995) was used for electrostatics, and LINCS (Hess, 2008) was used to constrain all bond lengths. The simulation data is available from the Zenodo repository (Ollila, 2018a).

The analysis and interpretation of 15N spin relaxation times are described in detail elsewhere (Ollila, Heikkinen & Iwaï, 2018). Briefly, rotational correlation functions of the backbone N–H bonds were calculated from the simulation data and the spin relaxation times were calculated using the Redfield equations (Abragam, 1961; Kay, Torchia & Bax, 1989; Ollila, Heikkinen & Iwaï, 2018). Before the spin relaxation time calculation, the overestimated overall rotational diffusion in the MD simulation due to the water model was corrected by dividing the rotational diffusion coefficients around all inertia axes with a factor of 1.2, assuming that the protein rotates as an anisotropic rigid body. This scaling factor was found to be capable of reproducing the 15N spin relaxation times in good agreement with the experimental data of PaTonB-96 for tip4p water model (Ollila, Heikkinen & Iwaï, 2018). The computer codes used for the analysis and the related data are available (Ollila, 2018b, 2018c).


NMR solution structure of PaTonB-96

The protein consisting of the C-terminal 96 residues of TonB from P. aeruginosa (PaTonB-96) was previously identified as a minimal domain that was soluble when expressed using an E. coli expression system, while other shorter fragments were not soluble even with several different fusion partners (Guerrero, Ciragan & Iwaï, 2015). This fragment has 45% identity (40/88 residues) to EcTonB-92, suggesting a similar structure to EcTonB. [1H, 15N]-HSQC spectrum of PaTonB-96 shows the well-dispersed NMR signals for amide groups (Fig. 2), indicating a globular folded conformation. All of the main-chain chemical shifts were assigned except for S244 and HN of H245. 95.2% of the expected side-chains were assigned. The assigned chemical shifts were used for the automatic analysis of NOE peaks with the CYANA to calculate the NMR structure. Figure 3 shows the lowest energy solution NMR structure of PaTonB-96 with the secondary structure elements and a superposition of the 20 lowest energy conformers. The structural statistics of the 20 NMR conformers are summarized in Table 1. PaTonB-96 adopts a mixed α/β structure with the topology of βαββαββ. The structure consists of a β-sheet composed of three anti-parallel β-strands (β2, β3, and β5), a β-sheet composed of two short β-strands (β1 and β4) and two short α-helices (αI and αII). The structural coordinates and the chemical shifts have been deposited in the Protein Data Bank (, PDB code: 6FIP) and BMRB (, accession number: 34235).

Two-dimensional [1H, 15N]-HSQC spectrum of one mM PaTonB-96.

Figure 2: Two-dimensional [1H, 15N]-HSQC spectrum of one mM PaTonB-96.

The spectrum was recorded at the 1H frequency of 800 MHz at 25 °C. The residue number and single letter code for amino acid types indicate the assignments. Trp HNe, Gln and Asn side chain amide resonances are marked by “sc.”
NMR structures of PaTonB-96.

Figure 3: NMR structures of PaTonB-96.

(A) Ribbon drawings of the lowest energy conformer of the PaTonB-96 structures showing the secondary structure elements. (B) Stereoview of an ensemble of the 20 lowest energy NMR conformers. Red and blue color are used for α-helices and β-sheets, respectively. N and C indicate N- or C-termini, respectively. Figures are generated with PyMol (Schrodinger, 2015).

Comparison of the NMR structures between E. coli and P. aeruginosa

The primary and secondary structures of PaTonB-96 were compared with the structure of EcTonB-137 (PDB: 1XX3) (Peacock et al., 2005) (Fig. 4). The length of PaTonB-96 is similar to that of the globular part of EcTonB-137 (88 residues). Notable structural differences are observed around the C-terminal end. The C-terminal end of EcTonB-137 forms an anti-parallel β-strand (β6) with β5-strand to constitute a β sheet. In contrast, the C-terminal end in PaTonB-96 is unstructured and exhibits an extended flexible conformation as also seen from the spin relaxation data (Fig. 5). The difference can be easily explained by the shorter C-terminal end after the β5-sheet in PaTonB-96, being too short to form an additional β-strand. The shorter C-terminal end in PaTonB-96 is compensated by the longer loop between β4 and β5-strands with additional five residues, compared with the structured 88-residue region of EcTonB-137. Higher mobility of this longer loop is also confirmed by the 15N relaxation measurement and MD simulation (Fig. 5). Interestingly, the extended and disordered conformation of the C-terminal end in PaTonB-96 more closely resembles the crystal structures of EcTonB-92 (PDB: 1U07) or the structures from TonB/TBDT complexes (PDB: 2GRX and 2GSK) (Fig. 1). The interaction between β5-strand of TonB and the TonB box in TBDTs has been proposed to be essential for the TonB-mediated energy transfer (Pawelek et al., 2006; Shultis et al., 2006). Such interaction would be hindered by the additional β6-stand found in EcTonB-137. Thus, our results suggest that β5-strand is more accessible for the proposed interactions with TonB box in PaTonB-96 where the β6-strand is absent.

Comparison between PaTonB-96 and EcTonB-137.

Figure 4: Comparison between PaTonB-96 and EcTonB-137.

(A) The sequence comparison between PaTonB-96 and the last 90 residues of EcTonB-137 with the secondary structure elements. (B) Stereoview of the superposition of the 20 NMR structures of PaTonB-96 (gray) and residues 151–239 of the 20 NMR structures of EcTonB-137 (PDB: 1XX3, blue). N and C stand for the N- and C-termini, respectively.
MD simulation of PaTonB-96 and comparison with the experimental data.

Figure 5: MD simulation of PaTonB-96 and comparison with the experimental data.

(A) The comparison of the experimental and simulated 15N relaxation parameters and the internal motions presented by S2 and te obtained from the MD simulation. The regions for β-sheets, αI, and αII are highlighted in blue, purple, and orange, respectively. (B) A superposition of snapshots of the structure from the MD simulation trajectory indicating conformational fluctuations during the MD simulation. The residues with enhanced flexibilities are colored in yellow. The aI-helix with orientation fluctuations is colored in purple. Rotational diffusion coefficient values obtained from the MD simulation of the experimental spin relaxation data are, Dxx = 1.51 ± 0.01, Dyy = 1.72 ± 0.03, and Dyy = 3.79 ± 0.03 (rad2·107/s). These result in Dav = (Dxx + Dyy + Dzz)/3 = 2.3 ± 0.02 (rad2·107/s), τc = (6Dav)–1 = 7.2 ± 0.02 (ns), A = 2Dzz−(Dyy + Dxx) = 4.4 and R = DyyDxx = 0.2, where A is the axiality and R is the rhombicity. The overlay of the trajectories was produced by vmd (Humphrey, Dalke & Schulten, 1996).

Structural motions in PaTonB-96

Fast dynamics (picosecond to nanosecond) of proteins has been commonly investigated by measuring the spin relaxation times (T1, T2, and heteronuclear NOEs) of backbone 15N atoms (Jarymowycz & Stone, 2006; Van Den Bedem & Fraser, 2015). Here, we characterize the structural dynamics of PaTonB-96 by combining T1, T2, and heteronuclear NOEs for backbone 15N atoms and MD simulations (Ollila, Heikkinen & Iwaï, 2018). The measured spin relaxation times for PaTonB-96 are shown together with the MD simulation results (Fig. 5A). The good agreement with the experimental data allows us to use the MD simulation trajectory to interpret the timescales for the overall rotational diffusion (τc), effective correlation times for internal mobility (τeff), and order parameters (S2). The overall rotational diffusion coefficients around inertia axes (see the caption of Fig. 5) show that PaTonB-96 has high axiality (A = 4.4), but with a rather low rhombicity (R = 0.2) suggesting that the protein has an approximately ellipsoidal shape. The MD simulation analysis estimates τc = 7.2 ns for the timescale of average overall rotational diffusion. This is in line with 6.9 ns estimated from the T1/T2 values using the model-free approach (Kay, Torchia & Bax, 1989; Ollila, Heikkinen & Iwaï, 2018) as well as with the values in the literature for monomeric proteins with the similar molecular weight (Krishnan & Cosman, 1998). We thus conclude that PaTonB-96 is monomeric in solution as was the previously published NMR structure for EcTonB-137 (Peacock et al., 2005).

The results reveal that there are four regions with enhanced internal motions in PaTonB-96 (Fig. 5). The regions with notable conformational fluctuations are colored in yellow and purple in the overlaid snapshots from the MD simulation (Fig. 5). The first few N-terminal residues exhibit low order parameters and long effective correlations times related to the enhanced conformational fluctuations, suggesting that the N-terminal region is already the beginning of the flexible central region of the TonB protein that connects between TM region and CTD. The MD simulation revealed orientational fluctuations in αI helix (purple in Fig. 5B). This fluctuation also influenced the order parameters and effective correlation times of the neighboring residues in the MD simulation (Fig. 5A). However, the changes were not observed in the 15N spin relaxation analysis, presumably because the 15N relaxation is not sensitive to the time scale of the fluctuation. Residues 320–326 in the loop between β4 and β5-strands indicate some enhanced conformational fluctuations, which were also detectable by the 15N spin relaxation analysis and order parameters but does not affect the effective correlation times (Fig. 5A). The last five residues of the C-terminal end (residues 338–342) showed the enhanced conformational fluctuations, characterized with lower order parameters, long effective correlation times, and changes in the 15N spin relaxation rates, which are consistent with the experimental 15N spin relaxation data. The flexible residues at the C-terminal end are in close contact with the αI helix, indicating orientational fluctuations as described above.

Modelling of PaTonB and TonB box interactions

It is believed that TonB conveys the CM chemical potential to TBDTs by structural changes via the interaction between CTD of TonB and TonB box, a short stretch of peptide chain in the N-terminus of plug domain of TBDTs (Cadieux & Kadner, 1999; Pawelek et al., 2006; Peacock et al., 2005; Shultis et al., 2006) (Fig. 1A). In the crystal structure of EcTonB-93/BtuB complex, EcTonB orients the αI helix against the plug domain and forms a parallel β-sheet interaction with the TonB box of BtuB (Shultis et al., 2006) (Fig. 6A). We created a hypothetical model of a complex between PaTonB-96 and TBDT by superimposing PaTonB-96 to residues 153–233 of EcTonB in the crystal structure of the complex because no structure of TBDTs from P. aeruginosa is available (Fig. 6B). β5-strand (residues 226–231) of EcTonB-93 and TonB box of BtuB forms the parallel β-strands, of which interactions are mostly hydrophobic (Fig. 6C). The modeled structure of PaTonB-96/BtuB complex indeed resembles the conserved TonB box interactions observed with the EcTonB/BtuB complex, supporting that the modeled structure is plausible. The highly conserved Val10 in the TonB box is located next to phenylalanine in both TonB structures (Phe230 and Phe336 in EcTonB-93 and PaTonB-96, respectively), suggesting that this hydrophobic interaction might be critical for the TonB/TBDT complexes (Fig. 6C).

Models for interactions between TonB and TBDTs.

Figure 6: Models for interactions between TonB and TBDTs.

(A) The crystal structure of EcTonB-93 (cyan) bound to BtuB transporter (green, PDB: 2GSK). (B) A cartoon model of the complex of E. coli BtuB transporter and PaTonB-96 (gray) created by the superposition of CTDs of the TonB proteins. (C) Illustrations depicting the interactions between β5-strand of EcTonB-93 (cyan) or PaTonB-96 (gray) with the TonB box of BtuB transporter from E. coli (green). The consensus TonB box sequences based on 57 sequences from UniProtKB/Swiss-Prot (PROSITE: PS00430) is shown at the top. (D) Two hypothetical interaction models for PaTonB/TonB box from probable P. aeruginosa BtuB (Uniprot: Q9I473). White circles and dotted black circles indicate exposed and buried sides of the model, respectively.


We are interested whether the structural features found in TonB and TonB/TBDTs complexes from E. coli are shared among other Gram-negative organisms. In the modeled structure of PaTonB-96/EcBtuB complex, the positive charge at the beginning of β5-strand (Lys332 in PaTonB-96) could complement the negative charge of Asp6 in the TonB box, of which negative charge can be found in the half of the TonB box consensus motif from various Gram-negative organisms (Fig. 6C). This might suggest that the positive-negative charge interaction could play an important role in the specific recognition of TonB box. However, putative BtuB sequences from P. aeruginosa do not contain the exact consensus TonB box motif shown in Fig. 6C. Based on the observation from the model of PaTonB-96/EcBtuB complex, we searched a potential TonB box sequence in a putative BtuB sequence (PaBtuB) (Uniprot: Q9I473). We found a stretch of the sequence “DQVVTATR” (residues 29–36) at the N-terminal region of the possible plug domain region, with which PaTonB might interact, and hypothesized two interaction models (Fig. 6D). In the model A, we assumed Val32 in the PaBtuB as the highly conserved Val in the TonB box motif. In this model, the negative charge of Asp29 could be located in the vicinity of Lys332 and the sequence of “VVTA” is identical to the PaTonB-96/EcBtuB model. In the other model (model B), we considered Asp29 as the key residue in the alignment. The highly conserved Val in the TonB box is now replaced by Ala in this model, but the negative charge at Glu339 in PaTonB could be better compensated by Arg36 in PaBtuB if the extended the last β-sheet of PaTonB.

It is noteworthy that similar interactions are not plausible for the solution NMR structure of EcTonB-137 due to the steric hindrance by the presence of an additional β6-strand. The β6-strand needs to be disrupted and exchanged to the TonB box for the similar interaction as observed in the crystal structures of the two TonB/TBDT complexes (Pawelek et al., 2006; Shultis et al., 2006). The solution NMR structure of PaTonB-96 elucidated in this work is accessible to the TonB box without the strand exchange, because the disordered and extended C-terminal end does not interfere with the interaction site. This open conformation at the C-terminus is also observed in the NMR structures of HpTonB-92 (Ciragan et al., 2016) and TonB-like protein, HasB from Serratia marcescens (Amorim et al., 2013). Thus it might be a more common structure among CTDs of TonB proteins across different organisms.

Our hypothetical model of PaTonB-96 interactions with the TonB box and crystal structures of TonB/TBDT complexes suggests that αI–helix positions toward the plug domain located in the TBDT barrel (Fig. 6A) (Pawelek et al., 2006; Shultis et al., 2006). It was previously proposed that the positively charged Arg166 residue in αI–helix of EcTonB-CTD could interact with the negatively charged Glu56 in plug domain of FhuA receptor and disrupt its structure, thereby lowering the energy barrier required for the pore opening (Pawelek et al., 2006). Interestingly, the MD simulation detected orientational fluctuations of αI–helix in PaTonB-96 (Fig. 5), which also contains positively charged Arg271. Such fluctuations might further facilitate the proposed disruption due to the interactions between Arg271 in PaTonB and the negatively charged residues in the plug domain of TBDTs. The flexible loop between β4 and β5 strands found in PaTonB-96 is located distantly from BtuB (Fig. 6B). Therefore, the modeled complex of PaTonB/BtuB does not directly indicate any structural role for this flexible loop.


We report the solution structure of PaTonB-96, which is largely similar to the previously reported monomeric NMR structure of EcTonB-137 (PDB code: 1XX3) (Peacock et al., 2005) except for the C-terminal end. Whereas the C-terminal region of NMR structures of EcTonB-137 formed an additional anti-parallel β6-strand with β5-strand, the C-terminal end of PaTonB-96 has extended flexible conformation, which resembles the crystal structures of TonB proteins interacting with TBDTs (Pawelek et al., 2006; Shultis et al., 2006). The absence of the last β6 strand in PaTonB-96 structure suggests that the β5-strand is more accessible for the interaction with TonB box than in EcTonB-137, for which the strand exchange is required. Furthermore, the structural model suggests that the electrostatic interactions between PaTonB-96 and TonB box might be more favorable than for EcTonB. Based on the structural model, we identified a potential TonB box sequence in PaBtuB. We also speculate that the orientation fluctuations observed in the αI–helix of PaTonB-96 detected in MD simulation could lower the energy barrier of the suggested channel opening by disrupting the plug domain structure within the TBDT barrel when the C-terminus of TonB is bound to TonB box. The NMR structures and PaTonB-96 could thus guide further experimental analysis to unveil the structural basis of the mechanism of TonB-dependent energy transduction.

Supplemental Information

Supplemental File.

DOI: 10.5412/peerj.5537/supp-1