changes compared to structures of smaller TonB fragments
6.4.1 Description of experimental structure
The overall size of the molecule is ∼ 50 ˚A by 20 ˚A by 20 ˚A. The structure of TonB92 presents secondary structure elements in the order of α∗ -β1-β2-α-β3 (Fig. 6.2). Strands β1, β2 and β3 associate to a three-stranded β-sheet.
The C-terminal strand β3 is longer than β1 or β2 and interacts with the corresponding part of a second TonB92 molecule by forming an intermolec-ular antiparallel β-sheet. Dimerization leads to a non-crystallographic 2-fold symmetry (Fig. 6.1).
Near the N-terminus a segment of eleven residues (“ES1”, Arg154-Pro164) containing the Gln160 region possesses backbone conformation close to
β-7See www.biozentrum.unibas.ch/personal/jseelig/AUC/software00.html
Figure 6.1: Three-dimensional structure of the dimeric TonB92 in ribbon re-presentation. One molecule is shown in red, and the other one is blue. The aromatic residues forming four aromatic clusters are shown in ball-and-stick representation. The C-terminal β-strand forms an antiparallel β-sheet with the other TonB92 molecule.
sheet except for one residue, Leu156. A short helix (“α∗”) formed by six residues (Ala165-Leu170) follows which is part of a loop reversing the direc-tion of the main chain. It is followed by a type I β-hairpin composed of β1 (residues Gly174-Val182) and β2 (residues Asp189-Lys197). The three residues PDG (Pro184-Asp185-Gly186) at the tip of the hairpin are a conserved motif for the Ton-B family and are a frequent sequence in three-residue β-hairpins [78] which confer high turn stability even in peptides [79]. After a further turn, helix α (residues Glu203-Met210) and another segment with conforma-tion close to β-sheet (“ES2”, Trp213-Glu216), which is apposed to part of ES1 (Arg154-Arg158) follow. Surprisingly only four hydrogen bonds of this interac-tion stabilize the conformainterac-tion of ES1: the main chains of Arg154, Leu156, and Ser157 form hydrogen bonds with the main chain of Arg214 and Glu216 (Fig.
6.3). Nevertheless, the electron density of residues in ES1 is well defined (Fig.
6.4), and their B-factors do not deviate from the average value. After β2, the main-chain again changes direction and forms the long C-terminal strand β3
Figure 6.2: Topological diagram of TonB92 showing secondary structure elements derived from the crystal structure. β-strands are indi-cated by arrows, and α-helices are indicated by cylinders. Struc-tural elements that were not observed in the structures of TonB77 and TonB85 are marked with an asterisk.
(Pro220- Thr236). β3 protrudes out of the domain by about 8 residues and associates with an antiparallel β30 of another molecule resulting in an inter-molecular β-sheet β3-β30.
6.4.2 Comparison with crystal structures of TonB77 and TonB85
In the following, the crystal structure of TonB92 will be compared with that shared by TonB77 and TonB85. The common structure of the latter two fragments contains the residues 165−238 and will be referred to as the “tight dimer.”
A superposition of one molecule from the crystal structure of TonB92 and one from the tight dimer (Fig. 6.5) and their secondary structure assignments (Fig. 6.6) show that the secondary structure elements β1, β2, α, and β3 are formed and arranged similarly in both structures. In the tight dimer, the two molecules are engaged by exchanging their β1-β2 hairpins with one another resulting in the formation of a six-stranded intermolecular antiparallel β-sheet
Figure 6.3: Ball-and-stick representation of the amino acid residues of TonB92, which are involved in stabilizing the extended N-terminal ES1 segment by formation of hydrogen bonds. The bonds are shown as dotted green lines with the distances given in ˚Angstroms (˚A).
The representation of the side-chain atoms is incomplete.
(see Fig. 5 of Ref. [60]). In contrast, in the TonB92 structure the β1-β2 hairpin does not exchange with another molecule but rather takes up the same place that is filled in the tight dimer with the β10-β20 hairpin from the other molecule. As seen in Fig. 6.5, the orientation of the β-hairpin of TonB92 and of the tight dimer can be superimposed onto each other very well. The comparison shows that the TonB chain has additional flexibility that is not obvious from the tight dimer structure and that results in two hinges before and after the β1-β2 hairpin. The backfolding of the β-hairpin weakens the monomer-monomer interaction and leads to both a reduced length of β1 in TonB92 and the formation of a new helixα∗(Fig. 6.6). Strandβ1 of the tight dimer starts with the residues Ala169, Leu170, and Arg171, whereas in case of
Figure 6.4: Electron density map (2mFo - DFc) at 3σ (dark blue) and at 2σ (light blue) around amino acid residue Gln160 of ES1. The side-chains of Asn159 and Gln162 show multiple conformations.
TonB92 these residues are part of the helix α∗. The presence of seven addi-tional N-terminal residues of TonB92 as compared with TonB85 thus seems to abolish the β1-β2 exchange leading to a remarkably different crystal struc-ture but retaining the basic arrangement of the secondary strucstruc-ture elements β1, β2, α, and β3 (see Figs. 6.5 and 6.6). In the tight dimer of TonB85, 10 N-terminal residues (Ala155-Pro164), previously shown to be important in in-teractions between TonB and the TonB box of the receptors [30], could not be modeled due to a high flexibility of this region. In the corresponding ES1 seg-ment of the TonB92 structure, residues 158-164 possess dihedral angles close toβ-sheet but lack any hydrogen bonds of the backbone and the side chains to other parts of the molecule. Interactions via four hydrogen bonds occur only further N-terminally between residues Arg154-Ser157 and ES2.
Conversely, in the tight dimer the residues of helixα∗ showβ-like backbone conformation and are positioned close to the ES2 residues to which they form
Figure 6.5: Superposition of the three-dimensional structures of one molecule of TonB92 (in red) with one molecule of the tight dimer i.e. the structure of TonB77 or TonB85 (in blue). The β1-β2 hairpin of the second molecule from the tight dimer is shown in yellow. The PDG-loop between β1 and β2 containing residues Pro184, Asp185, and Gly186 is indicated.
two hydrogen bonds. The interaction partners of ES2 thus shift by approxi-mately ten residues from the N-terminal end of ES1 in the TonB92 structure to its C-terminal end. These differences are surprising, because TonB77 and TonB85 both contain all residues which in the TonB92 structure form α∗and ES2 and in fact TonB85 even contains most of the residues that form ES1, except for one residue, Arg154.
Five aromatic amino acid residues were found to be conserved in TonB of several Gram-negative bacteria, forming four clusters in the tight dimer.
Point mutations of these residues lead to a reduced activity of TonB [80].
In the two different crystal structures, these aromatic residues cluster in the same way, and the residues even have similar orientations (Fig. 6.1). In particular the cluster composed of Phe180, Trp213, and Tyr215, respectively, can be superimposed upon the corresponding residues of TonB85 with low deviation (Fig. 6.7). Phe180 resides on the exchangeable β-hairpin. For this reason the aromatic cluster (Phe180, Trp213, and Tyr215) is formed by residues
Figure 6.6: C-terminal amino acid sequence of TonB from E. coli. The position of the N-termini of the fragments TonB92, TonB85, and TonB77 are indicated by arrows. The secondary structure elements found in the structure of TonB92 and the tight dimer (i.e. TonB77 and TonB85) are indicated by arrows (β- strands) and boxes (helices).
Conserved residues are shown with a black background. The seg-ments ES1 and ES2 are indicated by lines.
of both molecules of the tight dimer, whereas in the structure of TonB92 all three residues belong to one molecule.
In the other cluster of the tight dimer, residues Phe202 and Phe230 interact by stacking of their aromatic side chains. In the TonB92 structure a similar arrangement of these two residues is found but enlarged by Tyr163 from ES1.
The interaction of Phe202 with Tyr163 meets the criterion of an “edge on”
interaction [81] and thus may contribute to stabilizing the folding back of the β1-β2 hairpin in the two hinges beforeβ1 and afterβ2. Tyr163has been shown to be critical for FecA function [69].
The structure of TonB92 and the tight dimer share the formation of an intermolecular antiparallel β-sheet β3-β30. The residue pairing is, however,
Figure 6.7: Superposition of one cluster of aromatic residues, Phe180, Trp213, and Tyr215, of the TonB structures. The residues from the structure of TonB92 are colored in red. Residues of the tight dimer are given in blue and yellow depending on which of the two TonB molecules they belong to.
slightly different. In TonB92 the center is shifted by one residue toward the C-terminus compared with the tight dimer.