changes compared to structures of smaller TonB fragments
6.5.2 Is there a role for the two conformations in the transport process?transport process?
Experimentally two crystal structures are observed that also show a simple way to derive one from the other. It is possible that these obviously stable structures of the tight dimer and of TonB92 reflect two conformational states of native full-length TonB independent of whether the dimer actually forms in vivo or not. Given that, it could be that each state will play a role in two different, distinct moments of the transport process. Because the basic steps of that process are virtually unknown, only rather tentative hypotheses can be put forward.
Cross-linking data and experiments sensitive to molecular mass indicate that TonB binds the receptors at two different affinities, at a low affinity via a site in the C-terminal domain and at a higher affinity via a site further to the N-terminus. Among the subsequent associations, some likely are of higher affinity to dissociate the ligand from its micromolar binding site. The structure of TonB92 seems to be a good candidate for a conformation that binds to the unliganded receptor at low affinity, because the Gln160region of ES1 is surface-exposed. The TonB box may be suitably placed for the interaction only in the liganded receptor, which would explain the cross-linking results of BtuB and FecA with TonB [30, 69].
The association of ES1 with the TonB box could result in the dissociation of the weak interaction between ES1 and ES2 (which requires only four hydrogen bonds to be broken) and thus trigger a change to a conformation that resembles that of one molecule of the tight dimer. The β1-β2 hairpin with its rather stable PDG turn could fold out (see Fig. 6.5) and would render the C-terminal TonB domain a reactive protein toward the receptor due to the many unsa-turated hydrogen bonds. This could be the conformation of the C-terminal TonB domain in the complexes with its receptor. The dimer structure itself
might not form because of the smaller copy number of TonB compared with its receptors. Interestingly, a mutation of the Glycine in the PDG motif of the hairpin, G186D, abolishes growth on ferrichrome and confers strong resistance to colicins B and M (Table 3 in Traub et al. [86]), which supports the idea of an important role for the hairpin.
Other scenarios can be envisaged. Some of the proposed transport mecha-nisms involve a pulling force generated by conversion of chemiosmotic energy.
That force may apply to the N-terminus of TonB92 after binding to the re-ceptor and disrupt the ES1-ES2 bond. Subsequently ES1 may be pulled along ES2. Helix α∗ may unwind, and part of its residues eventually become ap-posed and hydrogen-bonded to ES2. The unwinding of α∗ would abolish the hinge, which bends the main chain backwards between ES1 and β1, and the β1-β2 hairpin would fold out.
The two conformations of TonB92 and of the tight dimer obviously are both rather stable, and their interconversion in solution may be inhibited by a rather high activation barrier. Khursigara et al. [70] describe dimer formation of the periplasmic part of TonB by FhuA. We speculate that liganded FhuA may act as a catalyst for TonB dimerization due to its interaction with the ES1 segment. Moreover, Khursigara et al. [70] and Brewer et al. [85] de-scribe a high affinity interaction site of TonB with FhuA further N-terminal of the C-terminal domain. After high affinity binding of one TonB molecule to FhuA the receptor may catalyze dimer formation of TonB if the latter is present at sufficiently high concentration. The result would be a heterotrimer formed by one liganded FhuA with a TonB tight dimer. After dissociation from FhuA, the TonB tight dimer would persist as observed by Khursigara et al. [70]. This could serve as an inactivation mechanism for TonB, prevent-ing TonB-dependent receptors from becomprevent-ing blocked by an excess of inactive periplasmic TonB domains.
In the above scenarios, the folding out of the β1-β2 hairpin is presumed to lead either to a transient but tight FhuA-TonB complex of 1:1 stoichiometry or to the formation of the tight dimer with an adjacent TonB molecule. An idea of the mechanism by which the former complex may arise may be provided by two structures of the related domain from TolA. This energy-transducing protein which belongs to the TolQ/R/A system is known to connect the
cy-Figure 6.8: Superposition of the three-dimensional structures of one molecule of TonB92 (red) and the C-terminal domain of TolA from E. coli (blue) (pdb accession code: 1Tol, Lubkowski et al. [59]). The N-terminal α-helix of TolA, which is not present in TonB, is not shown.
toplasmic membrane with the outer membrane [87]. The C-terminal domain of TolA has been crystallized as a monomer [39]. A superposition of TonB92 with the C-terminal fragment of TolA is given in Fig. 6.8 and shows a close superposition of the three β-strands and the longα-helix. A second structure shows this TolA domain complexed as a monomer with a bacteriophage coat protein, with which it interacts during infection [59]. The association shows an intermolecular β-sheet formed and stabilized by intermolecular hydrogen bonds between TolA and G3p.
At present, the details of the interactions and conformational changes that take place during energy-dependent transport involving TonB and its receptors remain far from clear. However, the two structures of the tight dimer and TonB92 represent constraints, which a final model of transport will have to take account of, and perhaps clues to the dynamic mechanism by which TonB interacts with the receptors and transduces energy to the transport process.
6.6 Acknowledgments
We thank the staff at the X06SA (Swiss Light Source/Switzerland) synchrotron beamline for their support. We are grateful to Ariel Lustig from the Biozen-trum Basel/Switzerland for performing the analytical ultracentrifugation of TonB92. We also thank Ramon Kanaster for assisting us in purifying TonB92 and Kinga Gerber for helpful discussions.
All living cells require energy, which is predominantly provided in the form of ATP Tri-Phosphate). The hydrolysis of ATP to ADP (Adenosine-Di-Phosphate) and phosphate delivers about 30 kJ/mol, which can be used by the cell [88, 89]. To generate ATP, cells utilize so called ATP synthases.
These are multisubunit protein complexes [90] (see figure 7.1). The ATP synthase can be separated into two subcomplexes. One is the soluble F1 part, responsible for ATP generation and the other one is the membrane
Figure 7.1: Model of the F0F1 -ATP-synthase (Image by R. L.
Cross [91]).
spanning F0 part. The latter makes use of the energetically favoured transport of protons (or sodium ions) across the cell mem-brane. It consists of one a- and two b-subunits and a ring of 10-14 c-subunits, the c-ring. The pro-tein structure of the F1 part was solved in 1994 by Abrahams et al.
[92] and gave impressing insights into the mechanism of ATP syn-thesis. However, the structure so-lution of the F0 part proved to be challenging due to the fact that it comprises the membrane spanning part of the ATP synthase.
Pending this PhD-thesis, we suc-cessfully solved the structure of the c-ring of a sodium dependent F-type ATP synthase from I.
tar-taricus, consisting of 11 monomers. Since the phase problem could neither be overcome by heavy atom derivatization nor by Se-Met substitution, we finally solved the structure by molecular replacement using a new software package called PHASER [93]. A low resolution (6 ˚A) electron microscopy model which was provided by Vonck et al. was used as search model.