The solution structure of the periplasmic domain ... - Wiley Online Library

Molecular Microbiology (2007) 66(4), 872–889

doi:10.1111/j.1365-2958.2007.05957.x First published online 10 October 2007

The solution structure of the periplasmic domain of the TonB system ExbD protein reveals an unexpected structural homology with siderophore-binding proteins Alicia Garcia-Herrero,1 R. Sean Peacock,1 S. Peter Howard2 and Hans J. Vogel1* 1 Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive NW Calgary, Alberta, T2N 1 N4 Canada. 2 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5 Canada. Summary The transport of iron complexes through outer membrane transporters from Gram-negative bacteria is highly dependent on the TonB system. Together, the three components of the system, TonB, ExbB and ExbD, energize the transport of iron complexes through the outer membrane by utilizing the proton motive force across the cytoplasmic membrane. The three-dimensional (3D) structure of the periplasmic domain of TonB has previously been determined. However, no detailed structural information for the other two components of the TonB system is currently available and their role in the iron-uptake process is not yet clearly understood. ExbD from Escherichia coli contains 141 residues distributed in three domains: a small N-terminal cytoplasmic region, a single transmembrane helix and a C-terminal periplasmic domain. Here we describe the first well-defined solution structure of the periplasmic domain of ExbD (residues 44–141) solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric structure presents three clearly distinct regions: an N-terminal flexible tail (residues 44–63), a well-defined folded region (residues 64–133) followed by a small C-terminal flexible region (residues 134–141). The folded region is formed by two a-helices that are located on one side of a single b-sheet. The central b-sheet is composed of five b-strands, with a mixed parallel and antiparallel arrangement. Unexpectedly, this fold closely reAccepted 31 August, 2007. *For correspondence. E-mail [email protected]; Tel. (+1) 403 220 6006; Fax (+1) 403 209 9311.

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd

sembles that found in the C-terminal lobe of the siderophore-binding proteins FhuD and CeuE. The ExbD periplasmic domain has a strong tendency to aggregate in vitro and 3D-TROSY (transverse relaxation optimized spectroscopy) NMR experiments of the deuterated protein indicate that the multimeric protein has nearly identical secondary structure to that of the monomeric form. Chemical shift perturbation studies suggest that the Glu-Pro region (residues 70–83) of TonB can bind weakly to the surface and the flexible C-terminal region of ExbD. At the same time the Lys-Pro region (residues 84–102) and the folded C-terminal domain (residues 150–239) of TonB do not show significant binding to ExbD, suggesting that the main interactions forming the TonB complex occur in the cytoplasmic membrane. Introduction Nearly all bacteria require micromolar concentrations of iron for optimal growth; however, the concentration of the free ferric ion in the crust of the earth or in the extracellular media of the host organisms is very low (< 10-9 M). Typically iron forms rather insoluble oxi-hydroxide complexes or it is tightly bound to proteins of the host organisms. Consequently, Escherichia coli and other related Gramnegative bacteria have had to develop very efficient ironuptake systems that allow them to overcome the poor availability of the free ferric ion in their environment (Braun, 1997; Clarke et al., 2001; Braun and Braun, 2002; Ferguson and Deisenhofer, 2004; Krewulak and Vogel, 2007). Many bacteria and fungi secrete siderophores which are low-molecular-weight ferric ion chelators that posses high selectivity and high affinity for Fe(III) (< 10-20 M) (Schalk et al., 2004). The siderophores are produced and secreted into the extracellular medium when the intracellular iron concentration is low and ferric iron needs to be transported into the cell. In addition, some bacteria can also utilize iron from the ironcontaining proteins of their host organisms, such as haemoglobin (Izadi-Pruneyre et al., 2006), haemopexin (Cope et al., 1998), transferrin (Mickelsen and Sparling, 1981) or lactoferrin (Perkins-Balding et al., 2004). The iron complexes are too large to directly enter into the cell

Solution structure of E. coli ExbD 873 and hence bacteria rely on dedicated iron-uptake protein systems in order to internalize them. These protein systems are generally composed of an outer membrane transporter (OMT) protein, a periplasmic binding protein (PBP) and a cytoplasmic ATP-dependent transmembrane transport system. The iron–siderophore complex is initially recognized and bound by extracellular loops of the OMT and with the aid of the TonB protein system it is moved into the periplasmic space where the PBP recognizes it and directs it to the cytoplasmic transmembrane cassette for import into the cytoplasm. Transport of the iron complexes through the OMT is thermodynamically unfavourable and the outer membrane possesses no obvious energy source to activate it. In this case, the energy required for the transport is provided by the TonB protein system which couples the electrochemical potential of the cytoplasmic membrane by transducing the proton motive force (Bradbeer, 1993; Braun, 1995; Larsen et al., 1999). This system is composed of three proteins: TonB, ExbB and ExbD (Higgs et al., 1998). Escherichia coli TonB is formed by 239 residues distributed in three distinct domains: an N-terminal transmembrane region (residues 1–32), an extended partially proline-rich region that can span a large part of the periplasmic space (residues 32–150) and a folded C-terminal periplasmic domain (residues 151–239). These three features can be recognized in all TonB proteins that have been uncovered through genome sequencing efforts (Chu et al., 2007). The structure of the isolated C-terminal periplasmic domain of E. coli TonB has recently been solved by nuclear magnetic resonance (NMR) spectroscopy (Peacock et al., 2005) and by X-ray crystallography (Ködding et al., 2005). Moreover, the interaction of this domain with different peptides resembling the ‘TonB-box’ region of various OMTs has also been studied by NMR and the OMT–TonB interaction interface of TonB has been mapped (Peacock et al., 2005). This work was recently extended through the crystal structures of two different OMTs in complex with the C-terminal domain of TonB (Pawelek et al., 2006; Shultis et al., 2006). The interaction of the OMT with TonB through the so-called ‘TonB-box’ region is thought to provoke a conformational change in the OMT that enables the release of the iron complex into the periplasm (Ogierman and Braun, 2003; Gumbart et al., 2007; Ma et al., 2007), although this structural change is not seen in the two aforementioned complex crystal structures. In contrast to TonB, the 244-residue ExbB protein is mostly embedded in the cytoplasmic membrane with three helical transmembrane segments (residues 16–39, 128– 155, 162–194). Only its N-terminal end (residues 1–15) as well as the small loop that connects the transmembrane segments 2 and 3 (residues 156–161) is located in the

periplasm. A larger loop that connects the first and second transmembrane segments (residues 40–127) as well as the C-terminal end (residues 195–244) is located in the cytoplasm (Kampfenkel and Braun, 1993a). Finally, the topology of the ExbD protein was first described by Braun and coworkers (Kampfenkel and Braun, 1992). It contains 141 residues distributed in three domains: a small N-terminal cytoplasmic domain (residues 1–22), a single hydrophobic transmembrane helix (residues 23–42) and a 98-residue C-terminal periplasmic domain (residues 43–141). Currently, no detailed structural information is available to understand the exact role of ExbD and ExbB in the bacterial iron-uptake process. However, it is believed that both proteins interact with TonB (Higgs et al., 1998) and work together to provide energy to TonB, but the exact mode of action is not yet clear (Chang et al., 2001; Postle and Kadner, 2003; Wiener, 2005; Chu et al., 2007; Ma et al., 2007; Postle and Larsen, 2007). Different mutants of ExbD and ExbB have been studied (Braun et al., 1996; Braun and Herrmann, 2004) and it has also been reported that the deletion of either the exbD or exbB genes generates the same phenotype (Fischer et al., 1989) where most of the TonB activity is lost. The residual activity of TonB detected in the absence of either ExbB or ExbD is attributed to the homologous TolQ, TolR system (Braun, 1989; Braun and Herrmann, 1993). It is also reported that ExbB and ExbD form multimers in the cytoplasmic membrane as in vivo cross-linking assays indicate that ExbB and ExbD can form homodimers and homotrimers (Higgs et al., 1998). Recent studies have shown that the molecular ratio of the three proteins in the cell is 1 TonB: 7 ExbB: 2 ExbD (Higgs et al., 2002), although this ratio does not necessarily represent the molar fraction of the proteins of the TonB complex in the cytoplasmic membrane. In this work we present the high-resolution threedimensional (3D) NMR structure of a monomeric form of the periplasmic domain of ExbD from E. coli. Our studies also suggest that the native state of the protein in vitro may be a multimeric form and by deuterating the non-labile protons and performing transverse relaxation optimized spectroscopy (TROSY)-based 3D NMR experiments the secondary structure of the multimeric state could also be determined. Finally, NMR interaction studies were performed to determine whether parts of the periplasmic portion of TonB could interact with the periplasmic domain of ExbD. Results Expression, purification and aggregation state analysis of the protein The ExbD periplasmic domain from E. coli carrying a C-terminal six-histidine tag was overexpressed in BL21

© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 66, 872–889

874 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel

Theoretical 1.0 mM H2O 1.0 mM H2O 0.2 mM H2O 0.2 mM H2O

pH 7.0 pH 3.0 pH 7.0 pH 3.0

Hydrodynamic radius Rh (nm)

Calculated number of residues

Multimer size

1.84 3.20 ⫾ 0.3 2.94 ⫾ 0.17 2.27 ⫾ 0.10 1.86 ⫾ 0.07

107 763 ⫾ 207 545 ⫾ 130 223 ⫾ 32 112 ⫾ 12

1 6–7 4–5 2 1

The hydrodynamic radius was calculated from the NMR diffusion data with the empirical equations given by Wilkins et al. (1999).

Protein resonance assignments and structure calculation Protein resonance assignments were obtained using standard strategies based on triple-resonance NMR experiments (Ferentz and Wagner, 2000). The protein was single and double isotope-labelled with 15N and 15 N,13C respectively (see Experimental procedures). The protein is stable for several weeks under the experimental conditions described earlier allowing us to perform all the 3D NMR experiments that are required for the determination of the protein resonance assignments. The analysis of these experiments yielded the assignment of almost

7

pH3 pH5 pH7

5 3 1 -1 -3 -5 -7 -9

-11 250

240

230

220

210

200

190

(DE3) E. coli cells and purified as described in Experimental procedures. In order to solve the solution structure of the periplasmic domain of ExbD by NMR in its monomeric form, it was important to verify that the protein would behave as such in the course of the NMR experiments. At a 1 mM protein concentration in 20 mM sodium phosphate buffer at pH 7.0 and 298 K the one-dimensional (1D) 1H spectrum of the periplasmic domain of ExbD indicated that the protein aggregates extensively under these conditions, and the line width of the signals is much wider than expected for a 12 kDa protein. In an attempt to reduce the extent of protein aggregation several experimental conditions were investigated. The protein multimeric size was monitored by NMR diffusion experiments where the hydrodynamic radius of a protein is calculated indirectly by measuring its relative diffusion rate compared with an internal standard (Wilkins et al., 1999; Weljie et al., 2003). The addition of up to 150 mM NaCl to the protein sample or different amounts of various detergents and organic solvents, such as glycerol, CHAPS, Triton X-100, methanol, ethanol or trifluoroethanol, or changes in the experimental temperature (298–310 K), did not result in any changes in the line width of the proton signals suggesting that they had no effect on the aggregation state of the protein. Surprisingly, when the protein was placed in low salt, unbuffered aqueous solution the signals slightly narrowed although under these conditions (1 mM protein concentration, low-salt aqueous solution, pH 7.0, 298 K) the NMR diffusion experiments indicated that the protein hydrodynamic radius corresponded to a highermolecular-weight oligomer. Another factor to consider is the pH of the solution: when it was gradually decreased from pH 7.5 to pH 3.0, the resolution of the spectra improved. Sharper protein signals were observed at pH 3.0 and NMR diffusion experiments confirmed that the extent of protein aggregation was reduced when the pH value was decreased; however, even at pH 3.0 the protein is still aggregated at 1 mM protein concentration. Finally, the concentration of the protein in the NMR sample was lowered to 0.2 mM, and under these conditions the line width of the protein is

that expected for a monomer. NMR diffusion experiments confirmed that at pH 3.0 the protein at 0.2 mM concentration is indeed in a monomeric state (Table 1). Circular dichroism (CD) spectra were recorded at different pH values in order to confirm that the global secondary structure content of the protein domain at pH 3.0 does not differ significantly from that at physiological pH. In Fig. 1A, superposition of the spectra recorded at three different pH values is shown; there are some minor changes in the CD spectra; however, the secondary structure calculated for the protein performed with the k2d program (Andrade et al., 1993; Merelo et al., 1994) does not vary significantly. The shape of the CD spectrum suggests that the ExbD domain is made up of a mix of a-helix and b-sheet motifs. These findings encouraged us to determine the solution structure of the monomeric form of the periplasmic domain of ExbD by NMR. The relatively low protein concentration that needed to be used in these experiments (0.2 mM protein concentration) was offset in part by the use of a cryoprobe giving enhanced sensitivity of detection.

Molecular Ellipticity *10-5 deg cm2 dmol-1

Table 1. The results of the NMR diffusion experiments.

λ(nm) Fig. 1. Circular dichroism spectra of the periplasmic domain of ExbD in low-salt aqueous solution recorded at pH 7.0 (black), pH 5.0 (dark grey) and pH 3.0 (light grey).


Solution structure of E. coli ExbD 875

106

L66

Fig. 2. 1H,15N HSQC spectrum for the periplasmic domain of ExbD from E. coli. The amide resonance peaks are labelled with the residue assignment. The spectrum was acquired at 700 MHz on a sample containing 0.2 mM protein in low-salt aqueous solution (pH 3.0) 10% 2H2O (v/v) at 298 K.

G96

T94 T109 T85

110 T82

N72

114

15N

(ppm)

G134 S52 T53/G84/T137 V81

G131

M116

E113 D99

118 N91

H123 D120

S54

T88

D83

I76

M119

E61

F104

D44 Y112

E136 D48 V133

126

N78 F103 R105

D111

9.5

9.1

A106

8.7

D79 I86

D107

L142 T101

A89

A140

I130 V110

L49

K62

L132 K69

V47

R59 A51

A70 Y65 K129

L115 A92

Q57

K97

K117

E135 E143 Y127 M74 V118

V45

K108 I76

A125 I28

K141

H

122

T121 K139

H

Q124

S67

L93

T55

H

T100 T114

E95

S54

L90 G77

S73

K98 K46

A138 I102

8.3 1H

G126

7.9

7.5

7.1

(ppm)

100% of the 1H, 15N and 13C resonances of the periplasmic domain of ExbD (Fig. 2). The structure calculation was based on the analysis of the nuclear Overhauser effects (nOes) observed in the 13C-edited and 15N-edited 3D-NOESY-HSQC experiments. A total of 2307 distance constraints derived from these experiments were included in the structure calculation. Additionally, a dihedral angle constraint file for the f and y backbone dihedral angles was generated with the TALOS program using the chemical shift list as input file (Cornilescu et al., 1999). A total of 112 predictions were introduced as angle constraints in the structure calculation. Finally, the hydrogen-bonding pattern of the protein was identified by using a combination of data that included: the identification of the secondary structure elements through the Chemical Shift Index (CSI) analysis (Wishart and Sykes, 1994), the assignment of the longrange nOe cross peaks and the identification of the amide groups with slow 1H,2H exchange rates using deuterium exchange experiments. According to these data, a total of 32 hydrogen bonds were identified and were introduced as additional distance constraints in the latter part of the structure calculation. The protein structure was solved with automated NOESY cross-peak assignment using the CANDID

algorithm implemented in the program CYANA 2.0 (Guntert et al., 1997; Herrmann et al., 2002). The calculation resulted in the generation of a bundle of 20 low energy structures based on 959 meaningful interproton upper distance limits and 112 f and y torsion angle restraints. The average target function of the bundle of 20 lowest energy structures was 0.27 ⫾ 0.08 Å, the root mean square deviation (RMSD) values of the folded region (residues 63–135) were 0.48 ⫾ 0.07 Å for the backbone atoms and 1.01 ⫾ 0.07 Å for the heavy atoms (Table 1). The quality of the structures was analysed with the PROCHECK-NMR program (Laskowski et al., 1993), and more than 98% of the residues are located in the favoured regions of the Ramachandran space. Table 2 summarizes the details of the structure calculation statistics. Description of the solution structure The monomeric solution structure of the periplasmic domain of ExbD from E. coli is well defined and consists of three regions: an N-terminal tail composed of the first 20 residues (residues 43–63) with an extended flexible conformation followed by a well-defined folded domain (residues 64–133) and a smaller and also unstructured C-terminal tail that comprises the last eight residues


876 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel Table 2. Summary of the statistics for the NMR solution structure calculation of the periplasmic domain of ExbD from E. coli. Total number of cross peaks Upper distance limits: Total Short range, |i-j| ⱕ 1 Medium range, 1 < |i-j| < 5 Long range, |i-j| ⱖ 5 Torsion angle restraints H bond constraints Distance restraints violations (> 0.1 Å) Angle restraint violations (> 5°) Average target function value Average RMSD (residues 65–135) backbone RMSD to mean (Å) heavy atom RMSD to mean (Å) Ramachandran analysis (residues 65–135) Residues in most favoured regions Residues in additionally allowed regions Residues in generously allowed regions Residues in disallowed regions

2307 959 562 169 228 112 64 3 0 0.27 ⫾ 0.08 0.48 ⫾ 0.07 Å 1.01 ⫾ 0.07 Å 83.1 15.3 1.6 0.0

(residues 134–141). The fold of the structured region consists of two a-helices that are positioned on the same side of a single b-sheet (Fig. 3). The b-sheet is formed by five b-strands: the first b-strand (b1, residues 64–69) is located in the middle of the b-sheet and it is flanked by b2 (residues 73–76) oriented in an antiparallel manner and by b4 (residues 102–106) oriented in a parallel manner; b3 and b5 (residues 79–81 and residues 130–133 respectively) are located at the edges of the b-sheet: b3 is antiparallel to b2 and b5 is parallel to b4. The two a-helices, named a1 and a2, form a ~30° angle with each other, a1 (residues 83–94) connects b3 and b4 and a2 (residues 112–125) connects b4 and b5 (Fig. 4). The loop that connects b1 and b2 is not as well defined as the remainder of the structured region. Protein dynamics

fast movements. Interestingly, it can also be observed that the values of the residues of the loop that connects b1 and b2 are similar to those observed for the terminal ends confirming the existence of some flexibility in this loop which could have some functional relevance. Multimeric state The Rh values of the protein under different experimental conditions were obtained from NMR diffusion experiments using the equations described elsewhere (Wilkins et al., 1999). They were: 1.86 ⫾ 0.07 nm for the monomer and

A C

C

N

N

B

C

β4

C

β1

β1

β4 β2

β5

The dynamic properties of the periplasmic domain of ExbD were studied in order to confirm the different motional behaviour observed between the folded region and the N- and C-terminal ends in the NMR solution structure described earlier. Standard experiments for 15N relaxation measurements (Ishima and Torchia, 2000) were carried out, and T1 and T2 relaxation times as well as heteronuclear steady-state 1H,15N nOe values were calculated (see Experimental procedures). The nOe and T2 values of the folded region and the Nand C-terminal ends are clearly distinct confirming the presence of different motions in these three regions (Fig. 5). The well-defined core region presents nOe and T2 values that are representative of a non-flexible folded region with slow motions, whereas the ones determined for N- and C-terminal ends (regions 43–65 and 134–141 respectively) represent regions with high flexibility and

α2

α2

β2

β5 β3

β3

α1

α1

N

N

Fig. 3. Solution structure of the periplasmic domain of ExbD from E. coli. A. Stereo-view of the bundle of 20 conformers obtained in the last cycle of the CYANA structure calculation, the backbone heavy atoms of residues 64–133 were superimposed for minimal RMSD. The residues are coloured according to their secondary structure (red for a-helical, blue for b-stranded, dark grey for undefined secondary structure). The region 135–141 and the His6-tag have no regular structure and are not shown in this diagram. B. Ribbon diagram of the core domain (residues 64–133) of the conformer with the lowest CYANA target function. The positions of the N-terminal and C-terminal ends are indicated in the diagram.


Solution structure of E. coli ExbD 877 D83

T94

α1

α2

Fig. 4. Topology diagram of the periplasmic domain of ExbD from E. coli. Residue labels have been included at the beginning and the end of each secondary structure motif for better understanding of the diagram. Topology diagrams of the C-domain of FhuD (residues 175–270, PDB: 1efd), CeuE (residues 195–283, PDB: 2chu), TroA (residues 193–278, PDB: 1toa), BtuF (residues 156–240, PDB: 1nz2), and the N-domains of FbpA (residues 1–112, 224–275, PDB: 1d9v) and LivJ (residues 1–117, 254–318, PDB: 2lvi) are also represented for comparison. Circles represent a-helices and triangles represent b-strands. The small arrows indicate the direction of the backbone chains, while the top of the triangles indicates the direction of the b-strand.

A125

C

Y112 S73

V81 β2

β3

K69 β1

I76

D79

A106 β4

V64

ExbD

V133 β5

I102

I130

N

CeuE (2chu)

FhuD (1efd) 3

2

B

C

A

3

2

D

B

D

E

C

1

A

A

B

C

D

B

1

F

1

3

2

D

2

3

A

C

D

1

TroA (1toa)

BtuF (1nz2) LivJ (2liv)

6

FbpA (1d9v)

5

4

E

E C

A

N

4 3

D

C

A

B

N

5

D

1

1

6

B 3

2

2

2.94 ⫾ 0.17 nm and 3.20 ⫾ 0.30 nm for the multimeric forms at 1 mM protein concentration and pH 3.0 or pH 7.0 respectively, suggesting the presence of anywhere between four and seven copies in the multimeric state (Table 1). The value obtained for the 0.2 mM sample at pH 3.0 agrees with the Rh expected for a protein of 107 residues. The Rh values obtained from the dynamic light scatering (DLS) experiments also indicate the presence of multimers (3.1 nm for 1 mM concentration at pH 3.0 and 3.3 nm for pH 7.0), which would also correspond to a pentamer, hexamer or heptamer coinciding with the NMR results. Unfortunately, with this technique it was not possible to measure the Rh of the protein at 0.2 mM because of an insufficient signal intensity. The effect of aggregation on the solution structure of the periplasmic domain of ExbD was further characterized by comparing the 1H,15N HSQC spectra of the monomeric form (0.2 mM protein concentration in low-salt aqueous solution at pH 3.0) and the aggregated state (concentration higher than 0.2 mM and

pH > 3.0). The effect of increasing the pH on the amide chemical shift is relatively large, making it difficult to identify every peak by overlapping the spectra recorded at different pH values. On the other hand, the variation of the amide peaks can be monitored when the protein concentration is increased to 1 mM at pH 3.0; it can be observed how some signals disappear and others shift, while the rest do not differ from the monomeric form. The region between amino acid residues 104–116 is the most affected. The chemical shift perturbations were mapped onto the molecular surface of the NMR solution structure of the periplasmic domain of ExbD (Fig. 6). Interestingly, the amide protons that disappear upon aggregation are confined to a specific area of the folded region while the amide signals that are not affected by the concentration change are situated in the N-terminal flexible tail (not shown in Fig. 6) and the a1-helix of the folded region. The residues between these two regions experience chemical shift changes for their amide resonances upon aggregation.


878 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel Fig. 5. NMR relaxation data of the periplasmic domain of ExbD from E. coli in the monomeric state that identifies regions of structure with different mobility. Plots of 15N T1 (A) and T2 (B) and steady-state 1H,15N nOe (C) were measured at 0.2 mM protein concentration pH 3.0 and 298 K. The data show that the N-terminal region and the last eight residues at the C-terminus are highly disordered as well as the loop connecting b1 and b2.

A 1200 1000

T1 (ms)

800 600 400 200

600

140

135

130

125

120

115

110

105

100

95

90

85

80

75

70

65

60

55

50

45

0

B

T2 (ms)

500 400 300 200 100

80

85

90

95

100

105

110

80

85

90

95

100

105

110

140

75 75

135

70 70

130

65 65

125

60 60

120

55 55

115

50

45 1

50

0

C

0.5

nOe

0 -0.5 -1 -1.5 -2 140

135

130

125

120

115

45

-2.5

Residue

Resonance assignment of the multimeric states The combination of protein deuteration and TROSYdetection NMR experiments (Pervushin et al., 1997) provides for a valuable experimental approach to obtain high-quality NMR spectra of large molecules that otherwise would give rise to very broad resonance signals. The incorporation of 2H for all the non-labile protons in the protein decreases the T2 relaxation times, thereby decreasing the line width and increasing the sensitivity of the 3D correlation experiments (Gardner and Kay, 1998; Ishima and Torchia, 2000). Therefore, the periplasmic domain of ExbD was triple-labelled (2H, 15N, 13C) in an effort to characterize the secondary structure of the multimeric form of the protein and compare it with the

monomeric NMR solution structure described earlier. Electrospray ionization mass spectrometry (ESI-MS) spectra confirmed that 80% of the non-labile protons were replaced by deuterons (data not shown). Using a series of 3D-TROSY experiments (see Experimental procedures) we were able to assign a large portion of the backbone resonances of the protein residues at 1 mM concentration and pH 7.0 (Fig. 7). The peaks for some residues were unobservable under these experimental conditions. These were the same residues that disappeared from the HSQC spectra when the concentration is raised higher than 0.2 mM at pH 3.0. This indicates that the same homomultimer is formed when the pH is raised and when the protein concentration is increased.



C

C N

C N N

Fig. 6. Three different perspectives of the surface plot of the periplasmic domain of ExbD from E. coli are represented by three colours: red, yellow and cyan. The red areas represent the residues for which amide protons disappear in the 1H,15N HSQC spectrum while increasing the protein concentration higher than 0.2 mM. They represent the probable multimer interface; the yellow areas represent the amide protons that shift and the cyan areas represent the ones that do not change. The respective ribbon diagrams are also shown for clarity.

In order to compare the secondary structure pattern of the protein domain in the multimeric and monomeric state, CSI calculations were performed; CSI analysis predicts secondary structure motifs of a given protein based on the difference of the chemical shifts values of a given protein with the random coil ones (Fig. 8). The CSI analysis of the Ca and C′ carbonyl carbons of the monomeric and the multimeric forms of the periplasmic domain of ExbD confirm that the overall secondary structure of the monomeric form and the multimeric state are comparable; however, the secondary structure of the residues that are not observed cannot be characterized and subtle structural changes in this area may give rise to the slight differences observed in the CD spectra recorded at a different pH. ExbD–TonB interaction studies As ExbB, ExbD and TonB are known to form a complex with each other that is located in the cytoplasmic membrane of the cell, it seemed worthwhile to us to determine whether their periplasmic domains could contribute to the formation of this complex. We therefore studied

the interaction between 15N-labelled ExbD and two 14and 19-residue synthetic peptides that encompass the proline-rich domain of E. coli TonB: Glu-Pro (residues 70–83) and Lys-Pro (residues 84–102). As well we studied the capacity of ExbD to bind to the folded C-terminal domain of TonB, for which we earlier reported the solution structure (Peacock et al., 2005). The construct used for solving the TonB solution structure encompasses residues 103–239, of which the first portion 103–150 is flexible and unfolded in solution, while the latter part, residues 151–239, has an overall globular fold. We used the well-established NMR chemical shift perturbation strategy (Zuiderweg, 2002) to look into the binding and we studied the interaction for the monomeric protein. Our data show that if the pH is carefully maintained at pH 3.0, the addition of the Lys-Pro peptide and TonB C-terminal domain does not cause changes in the HSQC spectra of ExbD. On the other hand, the addition of the Glu-Pro peptide caused several peaks to shift in the HSQC spectra (see Fig. 9). When mapped onto the surface of ExbD there appear to be several sites on the surface of the protein as well as the unstructured C-terminal end that are involved in Glu-Pro


880 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel Fig. 7. 1H,15N TROSY-HSQC spectra of the deuterated periplasmic domain of ExbD from E. coli recorded at 1 mM protein concentration in low-salt aqueous solution at 298 K and (A) pH 7.0 and (B) pH 3.0. The assignments of the amide protons are shown. Peaks indicated by a circle had lower intensity than the contour level chosen to represent the spectra.

A 105

L66

G96 G126 T94 T85

109

113

N72 S52

T53

15N

(ppm)

T82

E84

117

N91

S73

D99

G77

L90

D83

H123

121 E76

E136

K139

G134 E61 R59 Q57

125

A92 M86

A140

A89

K46 A138

F103 Y65 K98

9.2

K97

V118

L122

L49

V64

N78

K141 Y127

V45 L142

T101

A125

D74

V47

A70 A5162

L93

E95 T121

T55

I87

T100

T88

S54

F75 D120

D107

V81

I102

8.8

8.4

8.0 1H

7.6

7.2

(ppm)

B 106

G96

L66 T94

T85 T82

110 G134

N72

(ppm)

114

G131

E84

S52

V81

15N

A138 N91 G77

118

D83

D99 I87 D120 H

F75

90

D44

T121

A125 Y127

L128

L122 E143 V47 L49

N48 T101 A140

A51

V118 A92

K97

M86

L142

A89

V46

V64 Y65 K62

126 K129

T100 L93

Q57

A70 N78

K139

K141

D79

R59

T73 E95

T55

E61 V45

H

I76 E136

122

T88

S54

H

H Q124

T53

D71

I102

F103 K98

9.2

8.8

8.4 1H

8.0

7.6

(ppm) © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 66, 872–889


(1)

(2)

6 5

Δδ(ppm)

A

Δδ(ppm)

4 3 2

5 4 3 2 1

-3

0 -1 -2 -3 -4 -5

-4

-6

1 0 -1 -2

140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

Residue number

Residue number

7

5

6

4

5

3 2

3

Δδ(ppm)

B

Δδ(ppm)

4 2 1 0

1 0 -1

-1

-2

-2

-3

-3

-4 140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

Residue number

Residue number

7

9

6

7

5

5

Δδ(ppm)

C

Δδ(ppm)

4 3 2 1 0

3 1 -1 -3

-1

-5

-2

-7

-3

-9 140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

140 138 136 134 132 130 128 126 124 122 120 118 116 114 112 110 108 106 104 102 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44

Residue number

β-sheet

Residue number

α−helix

Fig. 8. Comparison of the Chemical Shift Index graphs of the (1) a- and (2) carbonyl carbons of the monomeric (A) form of ExbD and the aggregated forms at 1 mM protein concentration and pH 3.0 (B) and pH 7.0 (C). A schematic view of the secondary structure motifs that are found in the monomeric solution structure is also provided for allowing comparisons.

peptide binding. The binding is in fast exchange on the NMR timescale, suggesting a relatively weak interaction. Discussion The results obtained from the NMR structural and dynamic analysis established that the solution structure of the periplasmic domain of ExbD from E. coli comprises three distinct regions: two flexible tails (20 and eight residues long respectively) with a well-defined structured region (64–133) located between them that is composed of two a-helices that are positioned on one face of a single b-sheet. The DALI server (Holm and Park, 2000) allows for comparison of 3D protein structures and the results are given as a function of the Z-score, which measures the statistical significance of a match between the query structure and an existing one, and values lower than 2 are not considered. The results of the DALI search performed with the folded region of the ExbD periplasmic domain as well as with the full-

length domain did show 630 matches but none of them with a Z-score higher than 5.8. The fold of the ExbD periplasmic domain is similar to the RNAse H-motif fold. The arrangement of the b-sheet and the two a-helices is the same; however, all the members of this fold contain an extra a-helix at the N-terminal end that folds on top of the b-sheet at the opposite side of the other two a-helices as well as one or more extra b-strands in some cases. According to the SCOP database this fold contains seven superfamilies of proteins. Also, after careful inspection of the query results we were able to recognize the following PBPs: ferric enterobactin-binding protein CeuE (Muller et al., 2006) from Campylobacter jejuni, ferric hydroxamate-uptake protein FhuD (Clarke et al., 2000), vitamin-B12-uptake protein BtuF (Borths et al., 2002), arabinose-binding protein AraF (Vermersch et al., 1991), leucine/isoleucine/valine-binding protein LivJ (Sack et al., 1989) and D-ribose-binding protein RbsB (Bjorkman et al., 1994) from E. coli with Z-score values of 4.6, 3.2, 2.8, 2.4, 2.4 and 2.3 respectively.


882 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel Fig. 9. Illustration of the interaction between the periplasmic domain of ExbD and the Glu-Pro peptide: A. 1H,15N HSQC spectra of 15N-ExbD periplasmic domain in the absence (black) and presence of the peptide in a 1:1 (red) and 1:2 (green) ratio. The assignment of the most affected residues are labeled. B. Ribbon diagram of the periplasmic domain of ExbD, the amide protons that experience the larger shifts by the interaction are represented in cpk and red. C. Surface electrostatic plot of the folded region of the ExbD periplasmic domain in the same orientation as the ribbon diagram. Red represents negative potentials, blue represents positive potentials and white represents neutral potentials.

105

A

107 109

T109

111

15

N (ppm)

113 T137

115

S73 E113

117

T114 D83

119

K139/141

121 L115 E136

123

L142

D44/Y112

125

A140

D78

127

A89

A138 D111

129 9.7 9.5 9.3 9.1 8.9 8.7 8.5 8.3 8.1 7.9 7.7 7.5 7.3 7.1 6.9 6.7 1

B

H (ppm) C

The structure of all these proteins encompasses two globular domains connected by different types of linkers. According to the topology of these two domains and of the linker, AraF, LivJ and RbsB are classified as Type-I PBPs; their globular domains possess a central b-sheet composed of six parallel b-strands and at least two a-helices on each side of the b-sheet, while the two lobes are linked by three b-strands (Fukami-Kobayashi et al., 1999). The DALI results indicate that the matching residues of the N-terminal domains of the Type-I PBPs present RMSD values of 2.5, 2.6 and 2.5 respectively; however, the topology of these does not match the topology of the periplasmic domain of ExbD described in this article (see topology diagram of LivJ in Fig. 4). On the other hand, FhuD, BtuF and CeuE belong to the Type-III PBPs that are characterized by a long a-helix connecting the two globular lobes

(Krewulak et al., 2004). The topology of the C-domain of BtuF differs significantly from that of ExbD as the central b-sheet is formed by only four parallel b-strands. Interestingly, the C-terminal lobes of FhuD and CeuE, which are both part of iron-uptake systems in Gram-negative bacteria, have almost the same topology as the periplasmic domain of ExbD. FhuD contains an extra a-helix and CeuE presents an a-helix and a b-strand connecting b2 and b3 as opposed to the loop found in the periplasmic domain of ExbD (Fig. 4). This difference is expected given that in ExbD there are only two residues between these b-strands. The topology of the zinc-binding periplasmic protein TroA from Treponema pallidum has also been analysed (Lee et al., 1999) because it is part of the TypeIII PBP group (Fig. 4) (Krewulak et al., 2004). This protein, like BtuF, lacks one antiparallel b-strand and the central


Solution structure of E. coli ExbD 883 b-sheet is composed of only four parallel b-strands. Ferric binding protein FbpA from Haemophilus influenzae (Bruns et al. 2001) is a Type-II PBP and because of its involvement in the iron-uptake process in bacteria we also compared it. However, the topology of the N-terminal lobe of this protein differs greatly from that of the periplasmic domain of ExbD. Its central b-sheet is composed of five parallel b-strands that are sandwiched by two a-helices on each side (Fig. 4). Taken together, it can be concluded that the topology of the periplasmic domain of ExbD is most similar to that of the C-terminal lobe of those Type-III PBPs that take part in the iron-uptake process in bacteria. This finding suggests an evolutionary relationship which could have biological implications that will require further study. However, it is unlikely that ExbD can directly bind siderophores, as the ExbD structure only represents one lobe of the bilobal siderophore-binding proteins and hence it does not have a complete binding pocket. The N-terminal flexible tail of ExbD connects the transmembrane domain with a well-defined structured region. The three models that attempt to explain the mechanism of action of the TonB system all require a certain degree of mobility of its components. On the one hand, the shuttle model (Letain and Postle, 1997; Postle and Kadner, 2003) assumes that TonB is embedded in the inner membrane in the ground state and leaves the membrane in an energized state in order to interact with the OMT. This model proposes that the ground state of TonB is energized through interactions with ExbD and ExbB, which also interact with the energized conformation of TonB and in doing so stabilize it. On the other hand, the propeller model (Chang et al., 2001) suggests that TonB is permanently associated with the cytoplasmic membrane and, by a rotational motion of the C-terminal periplasmic domain, it interacts with the OMT and also with the aid of ExbD and ExbB. In contrast, Wiener and coworkers (Chimento et al., 2005; Gumbart et al., 2007) have suggested that ExbB and ExbD can exert a pulling motion on the TonB protein and thereby dislodge part of the cork domain from the barrel of the OMT. This suggestion has found some support in recent experimental studies (Ma et al., 2007). It seems evident that in all cases some flexibility of the protein complex is needed to achieve productive interactions between the TonB system and the OMT. In the case of TonB, it has been reported that its prolinerich region (Brewer et al., 1990) as well as the following 50 residues of the periplasmic domain (Peacock et al., 2005) can act as spacers spanning the periplasmic space and may be unstructured and flexible. In the case of ExbB most of the protein residues are either embedded in the cytoplasmic membrane or located in the cytoplasm (Kampfenkel and Braun, 1993a), which would leave the flexibility requirements of the ExbD–ExbB complex in the periplasm to ExbD. The NMR structure of the periplasmic

domain of ExbD presented here includes a 20-residuelong flexible region that allows for mobility of the welldefined C-terminal structured region of this domain with respect to the ExbB-containing membrane surface that would facilitate the interactions required to energize TonB. The data obtained in the NMR diffusion experiments and in the DLS measurements revealed that the periplasmic domain of ExbD aggregates extensively forming homomultimeric complexes at high protein concentrations in vitro. Even though the multimeric states described here do not directly reflect the in vivo conditions, in general these observations demonstrate the tendency of the domain to oligomerize and support the previous findings that ExbD forms homodimers as well as homotrimers (Higgs et al., 1998). It is also consistent with the proposal that the intact TonB system contains more than one ExbD molecule (Higgs et al., 2002). Our NMR diffusion data obtained at pH 7.0 and 0.2 mM protein concentration in fact reveal the presence of a dimeric species in solution, which may resemble the one produced by in vivo crosslinking (Higgs et al., 1998) (see Table 1). The multimeric form of the protein observed at higher protein concentrations is unlikely to exist as such in vivo; it is most likely a symmetric structure as no new amide peaks are detected in the 1H,15N HSQC spectra of the multimeric forms as would be expected in an asymmetric multimer. Our ExbD–TonB NMR interaction studies provided evidence that the Glu-Pro region of TonB (residues 70–83) can bind to ExbD, albeit weakly. The fast-exchange conditions observed in the NMR experiments suggest a KD value of about 10-4 M or higher. The Lys-Pro region (residues 84–102) and the folded region (residues 150–239) of E. coli TonB did not show significant binding to the periplasmic domain of ExbD. However, it should be stressed that these results were obtained at pH 3.0 for the monomeric structure while for the multimeric structure at pH 7.0 no interactions could be detected (data not shown). Thus, it appears that in vivo the ExbB–ExbD– TonB complex is mostly held together through contacts between the membrane portions of these proteins. As we have discussed, the C-terminal lobe of the siderophore-binding protein FhuD from E. coli has the same topology as the periplasmic domain of ExbD. Recently, Coulton and coworkers described the formation of a 1:1 complex between FhuD and TonB; they identified the interaction surfaces on both proteins by testing random phage peptide libraries and later confirmed this interaction in vitro (Carter et al., 2006). This could indicate that ExbD and FhuD could perhaps interact in a similar manner with TonB. However, the proposed binding surface for TonB on FhuD mapped to its ferrichromebinding pocket and not to the C-terminal lobe of FhuD; therefore, it seems likely that the fold homology is related to any TonB-binding properties of FhuD and ExbD.


884 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel

A Unconserved 0 1 2 3 4 5 6 7 8 9 10 Conserved . . . . . . . . . 10 E.coli_ExbD M A M H L N E N L D E.coli_TolR MAR ARGRGRR Consistency * * 3 2 2 4 4 4 2 2

. . . . . . . . DNGEMHDI D- - L K SEI * 0 0 13 36*

. E.coli_ExbD A E.coli_TolR D Consistency 2

. . ST AT 6*

. . . . . RP E KP NDNPP 42 4 3*

. E.coli_ExbD T E.coli_TolR F Consistency 2

. . . . . . EGKKDT KA NPKT 54 433 *

. . . . . STP- ESQAV 453 00 . T V 4

. 60 QP SS 4 3 . I F 4

110 . F F L I 4 4

. . . . RADK GGAK 2 4 2*

. 20 NV NI * 8

. . . . 70 VYLSV VI VEV * 364*

. . TP VP 4*

. F L 4

. . . K- SGI 40 0

. . . . 120 . . T VDYE T L DVPYD EI 3* 2* 6 37

. I L 7

. . DV DV * *

. . . ADN GQY 4 4 1

. . ML L L 7 * . S T 5

. . . . . MKV MD I KAL N 5 * 5 7 5

B

. 30 VL VL * *

. . 80 MFI V VV 5 38 . T L 3

. L L *

. I L 7

. I I *

. F F *

. . . . . 40 MVAAPL MAT API * 54* * 7

. . . . . GND- E KDRL 2 4* 00

. 130 . . . . . LH QAGYL LH S AGVK * * 4 * * 32

. . . - PV ERL 026

. 90 TD PP 32

. . AT I T 3 * . E E *

. . . . . . . V DVKVNL QSVEVDL 2 4* 5* 5*

50 P P *

. . . . . . . . 100 T MI T AL NAL QV VAEVSSR 3 5 8436562

. . . . 140 . . . KI GLV GE E SVGLM T QP 48* * 5 2 6 3

. T I 3

. . . . AKAK - - - 0000

C

Pred: Pred: CCCCCCCCCCCCCCCCCCCEEEEEECCCCCEEECCEECCH AA: DVKVNLPASTSTPQPRPEKPVYLSVKADNSMFIGNDPVTD 50 60 70 80

Conf: Pred: Pred: CCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCEEEECCC AA: QSVEVDLPDATESQAVSSNDNPPVIVEVSGIGQYTVVVEK 50 60 70 80

Conf:

Conf:

Pred: Pred: HHHHHHHHHHHHCCCCCEEEEECCCCCCHHHHHHHHHHHH AA: ETMITALNALTEGKKDTTIFFRADKTVDYETLMKVMDTLH 90 100 110 120

Pred: Pred: CCCCCCCHHHHHHHHHHHHHCCCCCEEEEECCCCCCHHHH AA: DRLERLPPEQVVAEVSSRFKANPKTVFLIGGAKDVPYDEI 90 100 110 120

Conf:

Conf:

Pred: Pred: HCCCCEEEEEECCCCCCC AA: QAGYLKIGLVGEETAKAK 140 130

Pred: Pred: HHHHHHHHHCCCCEEEEEECCC AA: IKALNLLHSAGVKSVGLMTQPI 140 130

Conf:

D DVKVNLPASTSTPQPRPEKPVYLSVKADNSMFIGNDPVTD 50

60

70

80

ETMITALNALTEGKKDTTIFFRADKTVDYETLMKVMDTLH 90

100

110

120

QAGYLKIGLVGEETAKAK 130

140

Fig. 10. (A) Alignment of the ExbD and TolR proteins from E. coli performed with the Praline alignment tool (Heringa, 1999; Simossis et al., 2005). Secondary structure prediction performed with the PSIPRED program (Bryson et al., 2005; Jones, 1999) of the periplasmic domain of ExbD (B) and TolR (C). Secondary structure motifs of the NMR solution structure of the ExbD periplasmic domain (D).

Braun and coworkers also demonstrated that the TolR and TolQ proteins can substitute in vivo to some extent for ExbD and ExbB respectively (Braun, 1989; Braun and Herrmann, 1993). These two proteins are part of the Tol protein complex which is involved in maintaining the integrity of the Gram-negative cell membrane. It is formed by the TolA, TolQ and TolR proteins that possess some

sequence (and structural) similarity with TonB, ExbB and ExbD respectively (Eickhelmerich and Braun, 1989). TolQ and TolR have the same membrane topology as ExbB and ExbD respectively (Kampfenkel and Braun, 1993b), while TolR and ExbD share 25% amino acid sequence identity (Fig. 10A). Although most of the conserved residues are in the hydrophobic transmembrane helical domain, the


Solution structure of E. coli ExbD 885 secondary structure prediction program PSIPRED predicts the same pattern of secondary structure for both proteins (Fig. 10B and C). We note that the theoretical prediction of the secondary structure of ExbD obtained agrees with the secondary structure pattern of the experimental NMR structure presented here (Fig. 10D). We have also cloned the periplasmic domain of TolR and overexpressed it. Preliminary NMR experiments indicate that this protein domain aggregates extensively under all experimental conditions tested which has so far prevented us from solving its monomeric structure. However, as the secondary structure prediction agrees with the structural data for ExbD, combined with the fact that the secondary structure prediction is the same for the periplasmic domains of both proteins, it seems that TolR will have the same overall fold as ExbD. Structural studies of the multimeric form of TolR are currently in progress, to further substantiate this notion. ExbD has been shown to be essential for the virulence of Phororahbdus temperate (Watson et al. 2005), and it is therefore a promising candidate for antibiotic development. Further studies into the potential quaternary structure of ExbB, TonB and ExbD and their interactions with other proteins will hopefully reveal targets for new classes of antimicrobials specific to Gram-negative pathogens.

For the deuteration of the non-labile protons in the protein domain the cells were gradually acclimatized to deuterated media by increasing gradually the 2H2O percentage in the media of the overnight cultures (0%, 30%, 60% and 100% 2 H2O). Once the cells grew overnight in the fully labelled and deuterated media a full litre of culture was inoculated and the protein was expressed and purified. The percentage of deuteration achieved was 80% of the non-labile protons (data not shown). All isotopes were purchased from Cambridge Isotope Laboratories and all the other chemicals were obtained through local suppliers.

TonB constructs The C-terminal domain of E. coli TonB (residues 103–239) was purified as reported previously (Peacock et al., 2005). Two synthetic peptides consisting of TonB residues 70–83 (Glu-Pro peptide: EPEPEPEPIPEPPK) and residues 84–102 (Lys-Pro peptide: EAPVVIEKPKPKPKPKPKP) were synthesized by Anaspec (San Jose, CA). The peptides were over 98% pure as determined by analytical high-performance liquid chromatography and mass spectrometry.

Circular dichroism The CD spectra were obtained at 298 K in a JASCO G-810 spectrometer; the samples contained 20 mM of protein in water at three different pH values. Ten spectra were recorded and averaged for every sample.

Experimental procedures Cloning, expression, deuteration and purification of the periplasmic domain of ExbD The DNA sequence encoding the periplasmic domain of ExbD from E. coli (residues 43–141) was cloned into the pET30a expression vector (Novagen). The protein construct is expressed under the control of the T7 promoter and contains two additional residues (LE) followed by a six residue histidine tag at the C-terminal end. The plasmid was transformed into competent cells of the E. coli BL21(DE3) strain for protein expression. In order to produce the unlabelled, 15 N-labelled, or 15N,13C-labelled protein the cells were grown in Luria–Bertani media, 15N-M9 minimal media, or 15N, 13 C-M9 minimal media, respectively, containing 30 mg ml-1 kanamycin. Protein expression was induced by adding 0.4 mM isopropyl 1-thio-b-D-galactopyranoside when the cells reached an OD600 of 0.5–0.7 absorbance units, followed by a 4–5 h incubation period at 37°C. The cells were then harvested, lysed using a French press and centrifuged. The protein was purified from the lysed cells using nickel affinity chromatography. The elution buffer was exchanged through extensive dialysis with water at pH 3.0 and the protein was concentrated with a Centricon filter (1 K cut-off) until the desired concentration was obtained. The protein was obtained in high yield (> 50 mg l-1) in the soluble fraction and a high degree of purity was achieved as confirmed by ESI-MS and SDSpolyacrylamide gel electrophoresis (> 99%).

NMR sample preparation and data acquisition The 2D and 3D spectra required for the structure determination were collected on a Bruker AVANCE 500 MHz spectrometer with a triple resonance inverse Cryoprobe with a single axis z gradient or on a Bruker AVANCE 700 MHz with triple axis gradient TBI probe. For the structure determination experiments, all the NMR samples prepared for these experiments contained 0.2 mM of single-labelled 15N protein or double-labelled 15N,13C in a 90% H2O/10% 2H2O mixture of low-salt aqueous solution at pH 3.0, and double-labelled 15 N,13C in 100% D2O adjusted at pH 3.0. In all cases 0.1 mM DSS (2,2-dimethyl-2-silapentane-5 sulfonic acid) was added to the samples for chemical shift referencing. Low-salt solution indicates that no additional NaCl, KCl or buffer was used other than the counter ions associated with the protein and those introduced while making pH adjustments. The program NMRPipe (Delaglio et al., 1995) was used to process the multidimensional NMR experiments and the program NMRView (Johnson and Blevins, 1994; Johnson, 2004) was used to analyse the spectra.

NMR diffusion experiments The 1D-NMR diffusion experiments were acquired on the Bruker AVANCE 700 MHz instrument. Protein samples that were lyophilized under the desired conditions were dissolved in 2H2O. The protein diffusion was measured relative to


886 A. Garcia-Herrero, R. S. Peacock, S. P. Howard and H. J. Vogel dioxane which was used as an internal standard (Jones et al., 1997; Wilkins et al., 1999; Weljie et al., 2003), Approximately 5 ml of a 1% solution of dioxane in D2O was added to the sample. Pulsed field gradient diffusion experiments were collected with the PG-SLED sequence. The gradient strength was incremented in 64 steps from 1.25% to 80% of the maximum output of the linear gradient amplifier and a total of 32 scans of 16 000 data points were collected at each gradient amplitude. The Bruker XWINNMR package version 3.0 was used to process the data, a 1 Hz line broadening value was applied before Fourier transformation, the decay of the peak intensities as a function of gradient strength was evaluated from the resulting series of spectra. Calculated values for the hydrodynamic radii were determined using a previously determined empirical relationship (Wilkins et al., 1999; Weljie et al., 2003).

NMR resonance assignments and hydrogen exchange measurements Backbone resonance assignments of the protein at pH 3.0 and 0.2 mM concentration were determined using conventional triple resonance experiments (Muhandiram and Kay, 1994), CBCA(CO)NH (Muhandiram and Kay, 1994), HNCBCA (Grzesiek and Bax, 1992), HNCO (Grzesiek and Bax, 1992; Schleucher et al., 1993; Kay et al., 1994; Stonehouse et al., 1995) and HN(CA)CO (Clubb et al., 1992). Side-chain chemical shift assignments were obtained by analysing the H(CC)(CO)NH (H)CC(CO)NH (Montelione et al., 1992; Bax and Grzesiek, 1993; Logan et al., 1993) and HBHA(CO)NH (Montelione et al., 1992; Bax and Grzesiek, 1993; Grzesiek et al., 1993) experiments. Backbone resonance assignments of the deuterated protein were obtained using the same strategy. TROSYenhanced triple resonance experiments were recorded and analysed: HNCO, HN(CA)CO, HNCACB and HNCOCACB (Salzmann et al., 1998; 1999; Eletsky et al., 2001). In all cases, in order to reference the proton chemical shifts the DSS resonance was assigned as 0 p.p.m. and 15N and 13 C chemical shifts were referenced indirectly to DSS. The backbone hydrogen exchange rate was measured by performing a deuterium exchange experiment. Immediately after dissolving a lyophilized 15N-labelled protein sample in 2 H2O, 1H,15N HSQC spectra are recorded every 30 min for 24 h.

(Guntert et al., 1997; Herrmann et al., 2002; Guntert, 2003) for automated assignment of the NOESY cross peaks. The CYANA 1.2 protocol consisted of seven iterative cycles of nOe assignment and structure calculation, starting from 50 randomized conformers, 12 000 torsion angle dynamics steps were performed per conformer in each cycle. The bundle of 20 conformers with the lowest Cyana target function of cycle 7 was chosen as representative.

NMR backbone dynamics, data acquisition and analysis For the determination of the 15N-longitudinal relaxation time (T1), the 15N-transverse relaxation time (T2) and the heteronuclear nOe’s NMR experiments were acquired at 298 K at 500 MHz (Ishima and Torchia, 2000). T1 experiments used longitudinal delay times of 5, 65, 145, 246, 366, 527, 757 and 1148 ms. T2 experiments used delays of 8.4, 27.8, 41.8, 58.6, 75.3, 108.8, 125.0 and 142.3 ms. The heteronuclear nOe was recorded with and without broadband 1H saturation using a saturation time of 3 s and a recycle delay of 6 s. Seventy-two scans per increment were collected with 1024 complex 1H points and 192 complex 15N points. T1 and T2 relaxation times were analysed by fitting the cross-peak volumes, measured as a function of the relaxation delay to a single-exponential decay in NMRView (Johnson and Blevins, 1994). Heteronuclear nOe values were calculated as the ratio of peak volumes in spectra recorded with and without saturation.

Acknowledgements This work was supported by an operating grant from the Canadian Institutes for Health Research (CIHR) to H.J.V. and an operating grant from the Natural Sciences and Engineering Research Council to S.P.H. A.G.H. was supported by a postdoctoral fellowship award from the Alberta Ingenuity Fund. H.J.V. holds a Scientist award from the Alberta Heritage Foundation for Medical Research (AHFMR). The NMR and CD equipment used was obtained through grants from the Canada Foundation for Innovation, the Alberta Science and Research Authority and AHFMR. Maintenance of the Bio-NMR centre is supported by CIHR and the University of Calgary. The structural co-ordinates for the periplasmic domain of ExbD have been deposited in the protein database under code 2pfu.

References Structure calculation For the structure calculation, proton distance constraints were obtained from the 3D 1H,15N HSQC-NOESY and 1H,13C HSQC-NOESY spectra (Palmer et al., 1991; Schleucher et al., 1994) acquired with a 150 ms mixing time. The cross peaks were manually picked and the peak volumes were determined with the NMRView automatic integration function (Johnson and Blevins, 1994). The TALOS program (Cornilescu et al., 1999) was used to generate a dihedral angle restraints file. The 3D solution structure was determined using the CANDID algorithm implemented in the program CYANA 1.2

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