The proline-rich domain of TonB possesses an extended polyproline II-like conformation of sufficient length to span the periplasm of Gram-negative bacteria
Silvia Domingo Kohler,1 Annemarie Weber,2 S. Peter Howard,3 Wolfram Welte,2* and Malte Drescher1*
'Department of Chemistry, University of Konstanz, Konstanz 78457, Germany 2Department of Biology, University of Konstanz, Konstanz 78457, Germany 3Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
DOl: 10.1 002/pro.345
Abstract: TonB from Escherichia coli and its homologues are critical for the uptake of siderophores through the outer membrane of Gram-negative bacteria using chemiosmotic energy. When different models for the mechanism of TonB mediated energy transfer from the inner to the outer membrane are discussed, one of the key questions is whether TonB spans the periplasm. In this article, we use long range distance measurements by spin-label pulsed EPR (Double Electron-Electron Resonance, DEER) and CD spectroscopy to show that the proline-rich segment of TonB exists in a PPII-like conformation. The result implies that the proline-rich segment of TonB possesses a length of more than 15 nm, sufficient to span the periplasm of Gram-negative bacteria.
Keywords: TonB; outer membrane; active transport; EPR; DEER; polyproline 11
Introduction ing of phages such as T1 (eponymous) and
625 and in some way transducing energy derived from In one model, TonB adopts an energy-rich con the proton motive force to them to effectuate formation through interaction with ExbBlExbD and transport. the proton motive force. This "loaded-spring" dissoci Within an N-terminal stretch of 32 residues (in ates from the cytoplasmic membrane, diffuses across E. coli) a hydrophobic segment anchors TonB in the the periplasmic space, binds to cargo-loaded recep cytoplasmic membrane (CM). This is followed by a tors and discharges its energy so that by means of proline-rich segment of ~100 residues that includes an unspecified mechanism the translocation of the a stretch of 68 residues in which more than 1 in ev cargo is effected ("shuttle model,,).22 In another ery 3 residues is a proline. A globular domain with model, TonB molecules, which are anchored in the affinity for TonB-dependent receptors forms the C CM but touch a loaded receptor on the periplasmic terminus of the protein. ExbB and ExbD, which ex side of the outer membrane, dimerize by means of hibit distant homology with components of the flag their C-terminal domain. ExbB and ExbD by means ellar motor/) appear to form a complex with of the proton motive force rotate the two TonB mole lO ll TonB. • Suppressor mutation analysis has sug cules. This torque leads to a twisting of the two gested that ExbB interacts with TonB through the TonB molecules pulling the C-terminal domains and transmembrane domain 12 while an NMR study sug the attached N-terminus of the receptor cork domain gested that ExbD interacts weakly with the periplas away from the outer membrane into the periplasmic mic domain. 13 space, thus unfolding the cork entirely or partially The crystal structures of several TonB-depend and abolishing the cargo binding site as well as cre ent receptors show a common architecture. 22- ating the exit channel ("propeller model,,).23 Gum stranded p-barrels span the outer membrane, with bart et al. 24 have studied the feasibility of partial the lumen filled almost completely by a cork domain unfolding or removal of the cork domain by a force formed by the N-terminal part of the receptor poly pulling at the C-terminal TonB perpendicular to the peptide. This leaves two pockets at the extracellular membrane into the periplasm by a molecular dy and the periplasmic openings of the p-barrel. Prior namics simulation. to TonB-mediated energy transduction, the cargo In accord with the shuttle model, the N-termi molecule is bound to a high-affinity site at the bot nal domain of TonB, which would be in the cyto tom of the extracellular pocket. Binding involves res plasm if it remained in the CM, could be labeled idues from the cork, the barrel and from the charac with a reagent impermeable to the cytoplasmic teristic long loops connecting p-strands of the barrel membrane, and only when it was functional in side on the extracellular side, which in some cases rophore transport.25 On the other hand, a study by enclose the binding site like petals. 14 The binding af Kaserer et al. 26 using TonB fusions is at odds with finity is typically in the nanomolar range, and bind the dissociation of TonB from the CM. For the pro ing occurs whether or not the cell has a functional peller model, the strongest support for the genera TonB system, indicating that energy transduction tion of a rotational torque stems from the homology through the TonB-receptor complex is required to between the ExbB and ExbD and the MotA and dissociate the cargo molecule from the binding site MotB proteins of the flagellar motor. However, no and to open an exit channel for its passage through evidence has been provided that TonB actually the barrel and into the periplasm. rotates and the observed 1:1 stochiometry of TonB Because TonB had been found critical for energy and receptor in the structure of the complex is not transduction from the cytoplasmic membrane to the in accord with this model without modifications. receptors, it was consistent that complexes of recep Thus far, differentiating between the shuttle tors with TonB were found to be stabilized upon and propeller models is not possible, in spite of the binding of cargo molecules. 15, 16 It was also in accord large amount of information about protein-protein with the observation of conformational transitions in interactions in the TonB energy transduction sys the structure of some receptors on the periplasmic tem. One item of information which could address 17 1H side after binding of cargo molecules. • Crystal the feasibility of the models, however, would be and NMR structures of the C-terminal globular do knowledge of the physical length of TonB. The width 15 19 main of TonB alone . and in complex with outer of the periplasm is estimated to be 15-20 nm,27 so 20 membrane receptors ,21 have been reported. In the TonB should match this length in order to be able to complexes the domain was in contact with the so deliver torque that is generated in the CM to a re called TonB-box of the receptors, a conserved stretch ceptor in the outer membrane. TonB and its homo of ~7 amino acids near the periplasmic N-terminal logues share a central proline-rich segment of 68 end of the cork domain. residues of which 24 are prolines. Moreover, in its Models have been proposed as to how TonB N-terminal half acidic residues but no basic residues might transduce electrochemical energy to the recep are abundant whereas in its C-terminal half this tors so as to effect cargo translocation from the re pattern is reversed. Short distance information from 28 ceptor binding site to the periplasm. NMR studies ,29 have already suggested that TonB
626 adopts an extended structure allowing it to span the estimated 20 nm width of the periplasmic space. The -- Ton8 59/69 1,0 anomalous migration in SDS-PAGE indicates rigid --Ton8 59/76 --Ton8 69176 ity and elongation as well. ~ o The high content of pro _. __. Ton8 69/84 c: line and the aqueous environment suggests that a o --Ton888/106 polyproline helix II (PPlI) would be a possible con S 0,8 --Ton8106/120 formation, that is a left-handed helix with an axial ~ translation of 0.31 nm per residue, rather than a I right-handed polyproline I helix (PP!) with an axial is 0,6 translation of 0.19 nm per residue. Experiments showed that the PPII conformation is maintained in proline-rich polypeptides up to a certain content of u other residues.: ,:l2 The dihedral angles of a PPII 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 conformation (cp = - 75", \~ = 145°, W = 180") resem t [)Is} ble those of an antiparallel ~-sheet. Both conforma tions are close to the most extended conformation Figure 1. DEER traces. Background corrected dipolar which a polypeptide can adopt in the absence of evolution data from four-pu lse DEER experiments for different double-Iabeled mutants of the proline-rich steric constraints. segment of TonB (thin solid lines). Thick solid lines In this study, we present circular dichroism correspond to the fit assuming a Gauss distribution, data as well as long range distance measurements parameters shown in Table I. for the proline-rich segment of TonB using pulsed EPR. EPR spectroscopy is a method for revealing structure and dynamics in disordered systems. EPR native TonB which are replaced by the cysteines, see techniques can access distances between 0.5 and 8 Figure SI in Supporting Information). nm measuring the dipole-dipole interaction between The protein conformation in aqueous solution two spins.33 Diamagnetic systems can be studied was trapped by shock-freezing, and the distance using spin-labels. measurements were performed with the frozen solu The results show that the proline-rich segment tion. To minimize spin relaxation due to proton of TonB exists in a PPII conformation with a length hyperfine interactions deuterated water was used as enabling it to span the periplasm of Gram-negative the solvent. Distances below 1.5 nm are accessible bacteria. On the basis of the conformational data by analyzing the broadening of continuous wave obtained here, we propose a modification to the pro (cw)-EPR spectra in frozen solution. In control peller model in which the mechanism of energy experiments, no differences between cw-EPR spectra induces a torque-generated mutarotation of the PPII of singly and doubly labeled TonB mutants were segment of TonB to a PPI conformation. obtained (data not shown). Therefore, intramolecular distances below 1.5 nm were excluded. Protein Results aggregationldimerization under the conditions used We first investigated the structure of the proline was also ruled out by analysis of DEER traces of a rich segment of TonB using a two-frequency, pulsed singly labeled mutant which showed a homogeneous EPR method [double electron- electron resonance three-dimensional spin distribution (see Fig. S2 in (DEER) or pulsed electron-electron double reso Supporting Information). nance (PELDOR)]. 33-37 The DEER method was cho The dipolar evolution curves obtained by DEER sen, because it allows the measurement of distances for the double-mutants are shown in Figure 1. Model from 1.5 nm up to 8 nm in disordered materials,s'1 free analysis revealed that the spin-label distance To obtain long-range information on the confor distributions could be well fitted by Gaussian distri mation of the central part of TonB, we expressed butions. To facilitate comparison we analyzed the and purified the proline-rich segment between resi DEER data assuming Gaussian distance distribu dues 56 and 126 of native E. coli TonB with a N-ter tions characterized by two parameters only. The ex minal Hexahis-tag (see Fig. SI in Supporting Infor perimental data could be fitted by this model (thick mation). Distances were then measured between solid lines in Fig. 1). Table I lists the parameters of pairs of spin-labels introduced by site-specific cyste these distance distributions for all of the mutants. ine mutagenesis and derivatized with the thiol reac The widths of the distributions observed do not tive spin-label MTSL (1-oxy-2,2,5,5-tetramethylpyr reflect the error of the method but the conforma roline-3-methyl-methanethiosulfonate). The following tional variability of the protein itself and of the six double cysteine mutants derivatized with MTSL spin-label linkers. Assuming a linker length of the were investigated: TonB 59/69, TonB 59176, TonB MTSL spin-label of ~ 0.5 nm the findings suggest 69176, TonB 69/84, TonB 88/106, and TonB 106/120 that the protein conformation is rather stiff but not (The pairs of numbers indicate the two residues of . completely rigid. To estimate the deviation from a
627 Table I. Parameters Characterizing the Gaussian Dis· may in fact be in a PPII conformation. As an inde tance Distributions Derived from DEER Measurements pendent assessment of this conformation, the circu Spin-labeled Distance Width of the lar dichroism spectrum of the purified unmodified residues (nm) distribution (nm) proline-rich segment of TonB was analyzed. The TonB 69/76 2.5 :+: 0.1 0.8:+: 0.1 results in Figure 2 show a prominent dip at 205 nm TonB 59/69 2.9:+: 0.1 0.8:+: 0.1 in the spectrum that is characteristic for the PPII TonB 106/120 3.3 :+: 0.15 1.4 :+: 0.1 conformation as shown using model systems. TonB 69/84 4.0 :+: 0.1 1.1 :+: 0.1 TonB 59/76 4.4 :+: 0.1 1.6 :+: 0.1 TonB 88/106 4.6:+: 0.1 2.1 :+: 0.15 Discussion Despite the wealth of information available concern ing the protein-protein interactions of TonB with linear backbone conformation a set of three double high affinity outer membrane receptors, the known mutants (TonB 59/69, TonB 69176, and TonB 59176) requirement for an energized membrane for trans was designed to allow for triangulation. Adding the port, the crystal structures of receptors, the C-termi distances found individually for both sections (59-69 nal domain of TonB and the complex of both, the and 69-76) results in 5.4 nm in total which is about mechanism by which TonB acts is unknown. The 20% longer than the distance of the 59-76 section shuttle and propeller models discussed in the litera measured directly, suggesting deviations from a lin ture entail radically different modes of action. ear conformation of the backbone. The assumption Because there is no high resolution structure of of a slightly flexible backbone is supported by the TonB in complex with ExbB and ExbD available and fact that the width of the distance distribution because of the dynamic nature of the envisaged increases with the distance between the correspond mechanisms, it is difficult to judge which of these ing spin-label pair. models is reasonable. A central tenet of the propeller The main value of the EPR distance measure model however is that TonB must be able to reach ments rests in the possibility of estimating the dis the receptor in the outer membrane, that is that it placement per residue of the proline-rich segment of can span the periplasm. The long range information TonB by adding the lengths of the individually concerning the structure of the proline-rich TonB measured sections. This resulted in an average spin segment obtained here via a spin-label approach label distance of 0.26 nm per residue and a full indicates for the first time that TonB is long enough length of 14.4 nm (see Table l). to span the periplasm. The total length of the differ To calculate the distance projected onto the helix ent sections of the proline-rich segment as measured axis from the distance between spin-labels attached by DEER and listed in Table I indicates a length of to a helical structure, one must take into account ~14.4 nm. In conjunction with an estimated 6.5 nm the distance due to the relative angular displace length for the C-terminal globular domain as meas ment of the two labeled residues. A rigid PPII helix ured on the crystal structure, a total length of more can be modeled as a cylinder with a diameter D of than 20 nm is conceivable (see Fig. 3). about 1 nm38 (see Fig. S3 in Supporting Informa The EPR data also indicated some flexibility of tion). Owing to the threefold point symmetry compo the protein conformation resulting in minor deflec nent of the PPII helix the additional distance contri tions from a stick-like conformation. Because of this bution is maximal when the relative angular displacement of the spin-labels corresponds to an angle of 120'. Using a linker length 1 of 0.5 nm the maximal additional distance contribution can be '0 estimated. For all spin-label positions under investi -15 ·500000 gation it is maxim ally 0.2 nm and on average 0.1 'E o nm and therefore can be neglected. Relative angular l·1000000 displacement could affect the triangulation experi >- ment as well. Here, the experimentally obtained dif 1,1500000 ference between the sum of the distances of both sec ill ~ tions and the distance in total is 1 nm. Relative :il. ·2000000 angular displacement corresponding to an angle of
0 120 would have the largest effect, but would result ·2500000+-~-,---~.--~.--~"-~'--~r-'---r-c--;r--~ in a difference of only 0.4 nm. In conclusion, the dif 180 190 200 210 250 260 270 ference obtained in the triangulation experiment Wavelength [nm] cannot be explained by angular displacement but Figure 2. CD spectrum of the proline-rich segment of indicates a slightly variable conformation. TonB. The CD spectrum of the TonB wild-type fragment The DEER results suggested that the TonB pro containing residues 56--126 shows the characteristic feature line-rich segment has an extended structure and of a polyproline-II-helical structure.
628 plasmic membrane flanked by the flagellum motor like proteins ExbB and ExbD. These proteins exert a torque on TonB resulting in a mutarotation if TonB is bound to the TonB-box of the outer membrane re ceptor resulting in an angular change of w from 1800 (trans) to 00 (cis) in the peptide bonds of the proline rich segment. The mutarotation leads to a partial or complete transition from the stable PPII to the unstable PPI conformation accompanied by a con 4,0 nm traction which can amount up to 40% of the length
2,9 Itlll of the proline-rich segment. This results in an inward force as is assumed for partial unfolding or Exb N · T~nllmal removal of the cork from the receptor by Gumbart B IM et al.,2,j thereby mediating dissociation of the cargo Figure 3. Scheme of the structure of TonS. Distances molecule from its binding site and translocation to derived from DEER measurements are indicated in the the periplasm. In future studies, we will examine diagram showing that the pro li ne-rich segment of TonS whether or not the proline-rich segment of TonB can spans the periplasm. [Color figure can be viewed in the undergo a transition from a PPII to a PPI online issue, which is available at www.interscience. conformation. wiley.com.] Acknowledgments backbone flexibility the individual spin-label distan The authors thank Bettina Nagele, Daniela Lehr, and ces of the TonB 59/69 and TonB 69/76 pairs were Frederike Eggers for experimental contributions as ~ 20% larger than the distance obtained directly well as Kay Diederichs for stimulating discussions. with the TonB 59/76 pair. The flexibility may facili tate the sampling by TonB of the OM for ligand References loaded receptors. The averaged spin-label distance 1. Wang CC, Newton A (1971) An additional step in the value was 0.26 nm per residue. Taking the backbone transport of iron defined by the TonB locus of Esche flexibility into account an averaged axial translation richia coli. J BioI Chem 246:2147-2151. of 0.31 nm per residue results. This is significantly 2. 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