Structural Asymmetry in a Trimeric Na+/Betaine Symporter, BetP, from Corynebacterium glutamicum

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J. Mol. Biol. (2011) 407, 368–381 Contents lists available at

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Structural Asymmetry in a Trimeric Na + /Betaine Symporter, BetP, from Corynebacterium glutamicum Ching-Ju Tsai 1 , Kamil Khafizov 2 , Jonna Hakulinen 1 , Lucy R. Forrest 2 , Reinhard Krämer 3 , Werner Kühlbrandt 1 and Christine Ziegler 1 ⁎ 1

Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany Computational Structural Biology Group, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany 3 Institut of Biochemistry, University of Cologne, Zülpicher Str. 47, 50674 Köln, Germany 2

Received 10 June 2010; received in revised form 10 December 2010; accepted 12 January 2011 Available online 31 January 2011 Edited by W. Baumeister Keywords: automated rigid-body fitting; electron crystallography; membrane protein structure; Na+-coupled transport; osmotic stress

The Na+ -coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake. © 2011 Published by Elsevier Ltd.

*Corresponding author. E-mail address: [email protected] Present address: C. -J. Tsai, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. Abbreviations used: 3D, three-dimensional; 2D, two-dimensional; NCS, noncrystallographic symmetry; EM, electron microscopy; LPR, lipid/protein ratio; TM, transmembrane; L-CC, Laplacian-filtered correlation coefficient; CC, cross-correlation coefficient; PDB, Protein Data Bank. 0022-2836/$ - see front matter © 2011 Published by Elsevier Ltd.

Introduction Under hyperosmotic stress, bacteria accumulate up to molar concentrations of organic molecules, such as betaine, in order to prevent dehydration of the cytoplasm. Since these so-called osmolytes, or “compatible” solutes, do not perturb protein function, such high concentrations are not detrimental to cellular function.1 For a rapid response to changes in osmotic pressure, specific tightly regulated membrane


Three-dimensional BetP Structure to 8 Å by EM

transport proteins are required to facilitate the uptake or extrusion of these compatible solutes. BetP is the main betaine transporter in Corynebacterium glutamicum that belongs to the betaine/ carnitine/choline transporter family. 2–4 BetP requires the cotransport of two Na+ per betaine molecule and can achieve extremely high substrate concentration gradients across the membrane of more than 6 orders of magnitude.2 The cytoplasmic ∼ 50-residue-long N-terminal and C-terminal domains of BetP are highly charged.5 Truncations at the N-terminal domain of more than 10 residues increase the osmolarity required for activation, while truncation of the C-terminal domain by more than 23 residues results in constitutive activation, albeit at a reduced level of activity.6 The role of the C-terminal domain in activation includes detection of the cytoplasmic K+ concentration, which acts as a measure of hyperosmotic stress.6 An important factor here may be that the Nterminal and C-terminal domains interact both with one another and with cytoplasmic loops. 7 In addition, BetP was shown to respond to stimuli originating directly from the membrane, such as changes in lipid composition. 8 In the entirely negatively charged lipid environment of C. glutamicum, BetP shows a shift to higher osmolarity required for activation compared to activation in Escherichia coli, which contains only ∼ 30% negatively charged lipids.8 A projection map of BetP from two-dimensional (2D) crystals grown in the presence of E. coli polar lipids and cardiolipin revealed a trimeric architecture of BetP in the membrane. However, significant differences between the three protomers indicated that the trimer does not have exact 3-fold noncrystallographic symmetry (NCS).9 We recently solved the structure of a regulated N-terminally truncated form of BetP (BetPΔN29) to 3.35 Å resolution by X-ray crystallography. 10 In these three-dimensional (3D) crystals, the BetP trimer also showed a break in 3-fold NCS around the osmosensing C-terminal domains, which form extended α-helices and are differently oriented in each protomer. To separate the roles of different conformational states within the trimer in regulation and transport, we have determined the 3D structure of a C-terminally truncated, constitutively active mutant of BetP (BetPΔC4511 ) at 8 Å in-plane resolution and 16 Å resolution perpendicular to the membrane by electron cryomicroscopy of 2D crystals. The 3D map clearly shows that BetPΔC45 is an asymmetric trimer in the membrane, like the wild-type protein. Rigid-body fitting of individual protomers in the electron microscopy (EM) map revealed an altered trimer architecture in the membrane compared to that found in 3D crystals. Furthermore, the conformations of the three protomers differ significantly from one another. By

comparison with structural data obtained for transporters sharing the same fold as BetP, at least two of the three distinct protomer states within the BetPΔC45 trimer could be assigned to different conformational states involved in the transport cycle of BetP.

Results and Discussion BetP is functional after 2D crystallization A deletion mutant of BetP truncated by 45 residues in its C-terminal domain (BetPΔC45) was used for the structure determination of membranereconstituted BetP by electron cryomicroscopy of 2D crystals. The C-terminal truncation in BetPΔC45 results in the deregulation of BetP in cells or proteoliposomes,6 rendering it constitutively active for betaine uptake even in the absence of osmotic stress, although its maximum activity is 25% of the optimal wild-type activity.6 Two-dimensional crystals were obtained by detergent dialysis9,12 both in the presence of E. coli lipid/cardiolipin and in native C. glutamicum lipids (Fig. 1a), respectively. Incubation of the 2D crystals with the substrate at betaine concentrations higher than 10 mM led to distortions in the 2D crystal lattice; therefore, crystals were grown in the absence of substrate betaine. To confirm that BetPΔC45 retains its functionality after 2D crystallization, we measured its [ 14 C] betaine uptake activity.13 Closed proteoliposomes were obtained by fusion of the 2D crystal sheets grown in C. glutamicum lipids with preformed E. coli liposomes. BetPΔC45 transports betaine in fused 2D crystals although the rate of transport by BetPΔC45 under these conditions is ∼ 15% less than that by BetPΔC45 reconstituted in proteoliposomes (Fig. 1b) at the same lipid/protein ratio (LPR). This deviation likely originates from subtle differences in the LPR of the two systems, since, for example, the vesicles may become leaky when the protein content is high. Indeed, uptake rates diminish with decreasing LPR in both proteoliposomes and fused 2D crystals (Fig. 1b). Thus, in spite of these small differences in maximal turnover, BetPΔC45 appears to be fully functional after 2D crystallization. We therefore proceeded to determine the structure of BetΔDC45 by testing two different lipid conditions, namely either an E. coli lipid/cardiolipin mixture or a C. glutamicum lipid extract. Two-dimensional crystals of BetPΔC45 grown in an E. coli lipid/cardiolipin mixture Well-ordered 2D crystals of BetPΔC45 occurred very rarely (in b1% of crystallization trials) in the presence of a 58:42 mixture of E. coli lipid/


Three-dimensional BetP Structure to 8 Å by EM

Fig. 2. Projection structure to 7.5 Å resolution of the 2D crystals of BetPΔC45 crystallized in the presence of a 58:42 mixture of E. coli lipid/cardiolipin. The map reveals four trimers of BetP in a rectangular unit cell of 186.6 Å × 167.0 Å. The trimers (orange broken outline) are related by P22121 symmetry. Protomers 1 and 2 have similar conformations, while protomer 3 appears to be in a different state. The red arrow indicates additional crystal contacts (red densities) between two protomers in symmetry-related trimers, adjacent to TM1 (blue densities) from each protomer.

Fig. 1. Functionality of BetPΔC45 after 2D crystallization. (a) Electron micrograph of BetPΔC45 2D crystal sheets stained with 1% uranyl acetate. (b) Dependence of betaine uptake rate by BetPΔC45 on protein concentration in proteoliposomes. Uptake rates at 0.64 Osm/kg are shown for three different LPRs (from 30:1 to 10:1) in 2D crystals (gray bars) and proteoliposomes (black bars). Error bars represent the standard deviation from three to five individual measurements. Inset: Micrograph of freeze-fractured 2D crystals of BetPΔC45 after fusion to preformed E. coli lipid liposomes at a final LPR of 20:1.

cardiolipin and under conditions that were previously successful for the 2D crystallization of wildtype BetP.9 The trimers (orange broken line) are related by P22121 symmetry, and a projection map could be calculated by merging three independent lattices to 7.5 Å resolution (Fig. 2). The map reveals four trimers of BetP in a rectangular unit cell of 186.6 Å × 167.0 Å. These trimers resemble the wildtype BetP trimer reported previously.9 Therefore, we can conclude that the asymmetric trimer previously observed for the regulated wild-type protein is also observed for the deregulated BetPΔC45 under similar conditions. However, in

this crystal of BetPΔC45, an additional density (Fig. 2, in red, red arrow) appears in the projection map between protomers 1 of neighboring trimers, which was not observed in crystals of the wild-type protein. Unfortunately, the yield of well-diffracting 2D crystals in E. coli lipid and cardiolipin was not sufficiently high for 3D data collection. Table 1. Electron crystallographic data for BetPΔC45 in 2D crystals Plane group Cell dimensions a, b, c (Å) α, β, γ (°) Number of imagesa Number of structure factors (IQ1–IQ4) Resolution limit for merging (Å) Effective resolution of 3D set (Å)b In-plane Perpendicular to the membranec Completeness (%) 0–50° 0–90° Overall weighted R-factor (%) Overall weighted phase residuals (°)d

p121_b 92.1, 155.2, 150 90, 90, 90 72 2677 8.0 8.0 16.0 87 73 29.4 17.9

a Nineteen at 0°, 5 at 10°, 22 at 20°, 14 at 30°, and 19 at 40–60° (nominal tilts). b As calculated from a point-spread function of the experimental data. c As calculated by the point-spread function. d From the program LATLINEK.

Three-dimensional BetP Structure to 8 Å by EM


Fig. 3. Data collection on 2D crystals by electron crystallography. (a) Plot of combined phase error to 7.5 Å merging 72 lattices. The number and the size of the boxes correspond to the phase error after averaging and rounding to 0° and 180° for each measurement (1, b 8°; 2, b 14°; 3, b 20°; 4, b 30°, where 45° is random). Values over 30° are indicated only with decreasing box size. The resolution limit was set to 7.5 Å. (b) Lattice line data shown as plots of amplitudes (bottom) and phases (top) along the z⁎-axis for the (4,1) and (3,5) reflections. The lattice lines were produced by weighted least-squares fitting, and resulting errors are shown.

Three-dimensional map of BetP in C. glutamicum lipids The 2D crystals of BetPΔC45 grown in the presence of primarily negatively charged C. glutamicum lipids reproducibly yielded images with structure factors to 8 Å and thus were used to calculate a 3D density map. The crystals belong to the p121-b symmetry plane group, with unit cell dimensions of 90.5 Å × 152.1 Å and γ = 90.8°. Seventy-two images of crystalline areas, obtained from specimens tilted by up to 50° under an electron

microscope, were processed and merged to 8 Å resolution with an overall phase residual of 17.9° (Table 1). The phases and amplitudes from fitted lattice lines (Fig. 3a and b) were combined to calculate the 3D map at an in-plane resolution of 8 Å (Fig. 4a and b). The resolution perpendicular to the membrane plane (assigned to the z-axis) was calculated to be 16 Å from the point-spread function of the experimental data. This reduced resolution in the z-direction reflects the cone of missing data that becomes prominent for images at high-tilt angles. To reduce the noise in the z-direction originating from

Fig. 4. Three-dimensional map of BetPΔC45 at 8 Å × 16 Å resolution. (a) View of one unit cell (92.5 Å × 155.2 Å). The three protomers of one trimer are labeled as protomers 1, 2, and 3. The broken line indicates the sectioning plane for the side view in (b). Three-dimensional densities are contoured at 1.3σ. (b) Side view of a cross section through the map indicated by the broken line pictured in (a) of the three protomers. (c) View of the density of individual BetP protomers 1, 2, and 3 contoured at 1.3σ from the top of the membrane. The protomers are aligned in the same orientation as protomer 1. The overall shape of protomer 1 is outlined by the broken line, which has been superimposed on protomers 2 and 3 for comparison. A red circle indicates the position of the putative substrate pathway. (d) An 8-Å density map constructed for a monomeric X-ray structure of BetPΔN29 (PDB entry 2WIT), superimposed on the structure. Repeat 1 is shown in red, repeat 2 is shown in green, and the peripheral helices TM1 and TM2 are shown in gray. Helices are labeled according to the domains to which they belong (i.e., where B signifies the bundle, H signifies the hash domain, and A signifies the arms connecting B and H). Thus, TM3–TM4 is labeled as B1–B2; TM8–TM9 is labeled as B3–B4; TM5–TM6 is labeled as H1–H2; TM10–TM11 is labeled as H3–H4; and TM7 and TM12 are labeled as A1 and A2, respectively. Note that the density here is computed to 8 Å in all three directions, which is the reason that densities can be observed for the helices that lie parallel with the membrane plane.

Three-dimensional BetP Structure to 8 Å by EM

Fig. 4 (legend on previous page)



Three-dimensional BetP Structure to 8 Å by EM

overweighting, we scaled down the amplitudes by applying a B-factor of − 300 Å2. At a cutoff of 1.3σ, the side view of the resulting map showed a singlelayered density with a moderate amount of noise perpendicular to the membrane (Fig. 4b). The three protomers (labeled 1, 2, and 3) are of similar overall size and shape (Fig. 4c, blue outline) and resemble the X-ray structure of a BetP monomer when displayed at a comparable resolution (i.e., 8 Å in all three dimensions) (Fig. 4d). A pore of ∼ 15 Å diameter is clearly visible in the center of each EM protomer, as well as in the X-ray density (Fig. 4c and d, red circle). At 3.35 Å resolution, this region is occupied by bulky aromatic side chains and by residues located in extramembranous loops,10 neither of which can be resolved at 8 Å. Many of the densities of the EM protomer maps appear to be difficult to reconcile with canonical transmembrane (TM) helices, which might at first glance appear to reflect poor-quality images or data. However, the low phase residual of 17.9° obtained for the relatively large number of images used argues against this (Table 1). In fact, these features reflect the complex overall fold of the protein and the arrangement of the helices therein. The BetP fold comprises a repeat of closely intertwined structurally related segments of five TM helices each (in Fig. 4d, the first repeat is in red, and the second repeat is in green). BetP shares this fold with transporters of five sequence-unrelated transporter families.10 The two TM5 repeats are related by a 2-fold pseudosymmetry axis running parallel with the membrane plane, such that they have opposite topologies with respect to the membrane. In the context of the structure, the first two helices of each repeat form a four-helix bundle (Fig. 4d, B1–B4), while an outer scaffold of helices surrounds the bundle. Within the scaffold, the third and fourth helices of each repeat form V-shaped elements that together create the socalled hash domain (Fig. 4d, H1–H4), and the last flexible helix of each repeat is assigned as arms (Fig. 4d, A1–A2). Such close intertwining of the helices causes their densities to merge, as is clearly seen for the four-helix bundle in the X-ray structure (Fig. 4d). Although such noncanonical densities are therefore expected for this fold and at this resolution, their assignment to specific helices is nevertheless a challenge; thus, to reduce the likelihood of misassignment, we carried out an automated fitting of the atomic X-ray structure to the EM density map. Automated fitting of the trimeric X-ray structure to the EM map Automated rigid-body fitting was performed using Situs14,15 in order to model the symmetric Xray trimer into the EM density of the BetPΔC45 trimer. This involved an exhaustive six-dimensional search with ∼ 1010 attempted fits. To achieve better

discrimination of correct fits between experimental maps and calculated maps, we took contour information into account using Laplacian filtering.14 The Laplacian-filtered correlation coefficient (L-CC) has a theoretical range of 0–1, although the absolute values tend to be significantly lower than standard cross-correlation coefficients (CCs). Nevertheless, seemingly small differences in L-CC can allow better discrimination of the fits than is possible with the standard CC.14 For the trimer, the seven highest-ranking fits deviate from one another only by small differences in tilt angle with respect to the membrane plane (the average difference between them is 3.9 ± 2.3°) and, in all cases, the X-ray structure overlays well with the EM density. The fit with the highest rank is shown in Fig. 5a and b. The L-CC scores of these top seven fits were between 0.090 and 0.085, and the corresponding CCs were 0.619–0.612. Fits in which the trimer is inverted with respect to the membrane plane were ranked lower (L-CC b 0.080 and CC b 0.590) and therefore excluded. Thus, we assign the top view of the 3D map of BetPΔC45 shown in Figs. 4 and 5 as from the periplasm. Automated rigid-body fitting of the X-ray structure to protomers in the EM map In none of the high-ranking trimer fits did the more perpendicular helices fit the map equally well in all three protomers simultaneously (e.g., while protomer 1 fits the density nicely, protomer 3 protrudes out of the membrane) (Fig. 5b). To account for those differences in protomer fits, we carried out automated rigid-body fitting of the X-ray protomer structure to each of the three densities in the 3D EM map. Automated fitting to the protomer maps was more challenging than for the whole trimer due to the reduced number of constraints. In particular, there was some ambiguity in the vertical positioning of these fits due to the limited resolution in this direction (see Materials and Methods). Nevertheless, for each protomer, we obtained one high-ranking fit (i.e., within the top 10) that was consistent with the general orientation in the trimer fit. The perpendicular helices in these protomer fits matched the regions of strong density better than in the trimer fits (Fig. 5c and d). For example, without further adjustment, peripheral helices TM1, TM6, and TM11 matched reasonably well with a set of three rod-shaped densities at the outermost corner of each protomer (Fig. 5c and d). The intensity of these densities is consistent with the perpendicular orientation of the peripheral helices in the X-ray structure with respect to the membrane plane. Based on these assignments, the central pore within each protomer of the 3D EM map (Fig. 5c, red circle in protomer 1) does indeed correspond to the substrate


Three-dimensional BetP Structure to 8 Å by EM

Fig. 5. Automated rigid-body fittings of the X-ray structure of BetPΔN29 to the 3D EM map of BetPΔC45. The EM density (blue) is contoured at 1.5σ. (a) Fit of the trimeric X-ray structure to the density of a trimer in the 3D EM map (viewed from the top). (b) View of protomer 1 (blue) and protomer 3 (green) from the plane of the membrane with the periplasm towards the top; the approximate membrane boundaries are indicated by parallel lines. Protomer 2 is shown in orange. (c) Fits of the protomeric X-ray structure of BetP to the density of individual EM protomer maps (viewed from the top) and (d) from within the plane of the membrane (with the periplasm towards the top). Helices in protomers 1–3 are color coded, and individual helices are numbered. The position of the substrate pathway is indicated with a red circle in protomer 1. Black arrows in the side views of (b) and (d) highlight the change in distance between the protomers on the cytoplasmic side due to a change in the tilt angle of the protomers with respect to the membrane plane.

pathway10 (Fig. 4c, red circle), which is lined by TM3, TM4, TM5, TM8, and TM10 in the X-ray structure. In addition, the strong off-center density adjacent to the central pore is occupied by the fourhelix bundle comprising TM3, TM4, TM8, and TM9; these helices are also oriented perpendicular to the membrane plane in the X-ray structure and therefore show up strongly in the EM map. Helices in the BetP X-ray structure—such as TM2, TM7, and the amphipathic helix H7 that run parallel with or adopt a shallow angle with the membrane plane— were only partly resolved in the 3D EM map, as mentioned previously. Finally, the densities for the tilted helices TM5 and TM10 (Fig. 5c), which connect

the hash domain to the four-helix bundle, are only partially resolved for similar reasons. The improvement from trimer fit to protomer fit reflects differences in tilt (10–15° for protomers 1 and 2, and ∼5° for protomer 3) relative to the trimer fit (Fig. 5b and d, black arrow). Nevertheless, significant differences between the three protomers are still clearly visible in, for example, the strong densities adjacent to the central pore, and these differences are observable at a wide range of σ levels (Fig. 6a). To test the extent of similarity between the protomer maps beyond their differences in tilt angle, we used the fitted protomer models to


Three-dimensional BetP Structure to 8 Å by EM

Fig. 6. Analysis of differences in densities in the three protomer maps. (a) Top view of a BetP trimer displayed at six different σ levels (1.4, 2, 2.5, 3, 3.5, and 4) showing that the differences between individual protomers are not caused by noise in the map. (b) Top view of the averaged EM model. Important features in the off-center density and at the perimeter are averaged out in comparison to the original EM map (left). Averaging could only be performed based on the individual protomer fits, as this allowed us to compensate for the different tilt angles with respect to the membrane plane.

average the EM density, resulting in a symmetric trimer (Fig. 6b). This process reinforced the densities of the scaffold helices TM5 and TM10, but weakened the strong off-center densities and the densities on the outer rim of the trimer, indicating that the protomers of BetPΔC45 are at least similar in the central region of each protomer and in the protomer–protomer interface. This result is consistent with the observation that the most pronounced differences between protomer maps are around the central pore and in the adjacent four-helix bundle (Fig. 4c and d). Therefore, we conclude that the asymmetric trimer of BetP reflects different conformational states of BetP. Optimization of BetP models to the three protomer maps To qualitatively assess the extent to which the three protomers differ, we adapted the models of BetP to make them more consistent with the densities in each protomer. For this, we used an automated flexible-fitting method involving rigidbody sampling of individual helix segments, followed by L-CC scoring (Materials and Methods). We focused our efforts on TM5 and TM12, which line one side of the central pore and are part of the scaffold, and on TM3 and TM8 from the four-helix bundle, which line on the other side (Fig. 5c). This process resulted in models that have higher (i.e., improved) L-CC scores compared to the rigid-body fittings of the X-ray crystal structure (Table 2), and that clearly differ from one another structurally (see,

e.g., TM3 in Fig. 7a and b). Furthermore, when each of the protomer models was scored against the other two EM protomer maps, the scores were consistently lower (Table 2), consistent with the differences between the models arising from optimization to a particular map and providing support for the proposal that the three protomers adopt different conformations. Comparison with conformational changes in a related transporter The conformational changes that BetP undergoes may well be similar to those proposed for Table 2. L-CCs of atomic structures fitted to the protomer maps of BetPΔC45 Models a



Protomer 1

Protomer 2

Protomer 3

Protomer 1 Protomer 2 Protomer 3

0.066b 0.125 0.072

0.092 (0.090)c 0.104 (0.119) 0.081 (0.079)

0.086 0.142 0.072

0.078 0.129 0.093

a For protomer 1, the scores of a second model are also shown (in parentheses). b The fit of the X-ray structure for this map was adjusted in the axis normal to the membrane (see Materials and Methods). c The L-CC for the highest-scoring model for each map is shown in boldface. For reference, the standard correlation coefficients (without Laplacian filter) for these fits are in the range of 0.43–0.49. Note that correlation coefficients for different maps cannot be compared due to differences in the intensities of density.


Three-dimensional BetP Structure to 8 Å by EM

Fig. 7. Different conformations of BetP described by automated flexible fitting to individual protomers of BetP and compared with two conformations of Mhp1. (a and b) The densities of BetP protomers 1 and 2 at 1.3σ, viewed from the periplasmic side and overlaid with the models generated by flexible fitting of TM3 (red), TM5 (yellow), TM8 (cyan), and TM12 (blue). The models are shown in spiral representation. The four-helix bundle is indicated by a red broken circle. TM4 (orange) and TM9 (dark cyan) of the bundle, and TM6 (yellow), TM7 (lime), TM10 (slate), and TM11 (blue) of the scaffold were not allowed to move during the flexible-fitting protocol. (c and d) Maps constructed at 8 Å resolution for (c) the outward-facing state of Mhp1 (PDB entry 2JLN) and for (d) the inward-facing state of Mhp1 (PDB entry 2X79), with the corresponding structures overlaid, and viewed in a similar orientation as the BetP protomers. The position of the fourhelix bundle (red broken circle) consisting of TM1 (red), TM2 (orange), TM6 (cyan), and TM7 (dark cyan) changes relative to the scaffold helices TM3–4 (yellow), TM5 (lime), TM8 (slate), and TM10 (blue).

Mhp1,16,17 as the two proteins share the same core fold. The proposed transport mechanism derived from structures and simulations of Mhp1 involves a rigid-body movement of the bundle and the hash domain (Fig. 4d) relative to each other, similar to the rocking-bundle mechanism first suggested by Forrest et al. in which the bundle of LeuT tilts relative to the scaffold.18 However, other mechanisms, such as flexing of the first helices of each repeat in the four-helix bundle, have been proposed.19,20 To further understand

the differences in individual protomers in the case of BetP, we compared their conformational differences (Fig. 7a and b) with those observed for different structures of Mhp116 at a resolution of 8 Å (Fig. 7c and d). The differences in conformation between an outward-facing open state [Protein Data Bank (PDB) entry 2JLN] and an inward-facing open state (PDB entry 2X79) of Mhp1 are clearly detectable after the conversion of the X-ray structures into 8-Å-resolution maps (Fig. 7c and d).

Three-dimensional BetP Structure to 8 Å by EM


Fig. 8. Different conformational states within a trimer of BetP. Flexible-fitting models viewed from within the membrane. The accessible substrate pathway in the center of each protomer is shown as a red surface. Tryptophan residues from TM8 (W362, W366, W373, W374, and W377) are shown as green spheres.

Specifically, differences in the relative position of the four-helix bundle consisting of TM1–TM2 and TM6– TM7 in Mhp1 (Fig. 7c and d, red broken circle) and the scaffold helices (TM3, TM5, TM8, and TM10; Fig. 7c and d) can be observed. In the outward-facing state, the bundle helices (e.g., TM1 and TM6) are more distant from the scaffold (e.g., TM3 and TM10) than in the inward-facing state, when viewed from the periplasm. The density in the region of the bundle also alters between these two states, changing from a solid density in the outward-facing open state of Mhp1 (Fig. 7c) to a ring shape in the inwardfacing state of Mhp1 (Fig. 7d). Importantly, the observed differences in those regions of the BetP EM maps are similar to those visible in the maps calculated for Mhp1. Thus, we are confident that conformational changes observed in transporters with the fold of BetP are indeed detectable in the protomer maps of BetP. However, a definitive assignment of the protomers to specific conformational states remains a challenge, even given different models from flexible fitting, because the differences in the L-CC scores of those models are relatively small. Instead, we rely on the comparison to Mhp1, based on which we suggest that protomer 1 adopts an outwardfacing conformation and protomer 2 adopts an inward-facing conformation (Fig. 7a and b). A subsequent comparison of the pathways in the models created by the flexible fitting of the X-ray structure to these two protomer maps is consistent with that assignment (Fig. 8). Protomer 3 is rather more difficult to assign but may also resemble an inward-facing state (Fig. 8). We note that at the current resolution, it is not possible to determine whether or not any of these states is substrate bound. Betaine was not added during crystallization; in any case, both the open state and the occluded state can exist in both apo

form and holo form during the transport cycle of a symporter such as BetP. In conclusion, although we refrain from a definitive assignment of the three protomers, the presence of different conformational states seems to be the most plausible explanation for the asymmetry in the BetPΔC45 trimer. Functional relevance of structural asymmetry in the BetP trimer Oligomerization of secondary transporters is generally assumed to occur for stability reasons, although specific evidence that it is (or it is not) functionally relevant is only available for a few transporters.21–24 Such questions are very difficult to answer, as demonstrated by the controversy over mitochondrial ADP/ATP carriers.23 In a crystal environment, if all protomers in an oligomer are able to function independently, every protomer can be expected either to adopt the same low-energy state or to show an average overall accessible state; both of these situations should result in a symmetric oligomer in the EM map. Indeed, most X-ray or EM structures of secondary transporters consist of symmetric oligomers. Exceptions are the structure of the small multidrug transporter EmrE from E. coli, which forms an asymmetric homodimer in 2D and 3D crystals,25,26 and the structure of the multidrug exporter AcrB from E. coli, which forms an asymmetric homotrimer in 3D crystals.27,28 In AcrB, the asymmetric oligomer state is thought to be functional, whereas the functional relevance of the asymmetric structure of EmrE remains controversial.29 Questions therefore arise: Why do BetPΔC45 and wild-type BetP trimers in 2D crystals exhibit significant asymmetry9 while the trimeric X-ray structure of BetPΔN29 appears more symmetric? 10 Which of these arrangements is physiologically more relevant?

378 One possible explanation for the observed differences between the X-ray structure and the EM structure of BetP is that they are due to the different constructs of BetP used. While the X-ray structure was determined from an N-terminally truncated mutant, the EM map was obtained from a C-terminally truncated BetP. Although both of these forms are functional active, they show differences in transport and regulatory properties. The N-terminal and C-terminal domains play an important role in the function of BetP:5–8,11 truncation of the C-terminal domain renders BetP constitutively active at a lower level, 6 while truncation of the N-terminal domain shifts the activation threshold of BetP to higher osmolalities so that the transporter becomes less sensitive to osmotic stress. 5,10 Moreover, mutual N-terminal and C-terminal interactions are important for betaine transport.7 However, a trimer containing two essentially identical protomers was observed in a projection map of BetPΔC45 2D crystals when the crystals were grown in a different, less polar lipid environment12 (Fig. 2). Based on the rigid-body fittings of the X-ray structure to the 3D EM map (Fig. 5), it appears that, in this crystal packing, contacts between trimers are maintained by the full-length N-terminal domain from a single protomer of each trimer. This is analogous to the situation observed in the symmetric X-ray structure of BetPΔN29, where only one of the C-terminal domains within the trimer is involved in crystal contacts.10 By contrast, crystal contacts between Nterminal domains are not observed in the 2D crystals of wild-type BetP, nor crystal contacts of BetPΔC45 in the presence of the C. glutamicum lipids shown here. In BetPΔC45 crystals, the C. glutamicum lipids most likely promote a particular crystal packing (p121) in which adjacent trimers are oriented towards opposite sides of the membrane and, thus, their terminal domains cannot interact. In these crystals, BetPΔC45 forms an asymmetric trimer with each protomer in a distinct conformation, like the wild-type protein. A dependence on the environment also explains the previously reported mirror-symmetric trimers in the 2D crystals of BetPΔC45 grown in a 58:42 mixture of E. coli lipid/cardiolipin.12 Apparently, this less negatively charged lipid environment places fewer constraints on the BetPΔC45 trimer, allowing the protomers to adopt different conformational states. Thus, the superposition of trimers during EM data processing led to a mirror symmetry12 similar to that obtained by averaging the different conformational states (Fig. 6b), presumably because the outward-facing and inwardfacing states are not in a fixed order in this lipid environment. In summary then, crystal contacts formed by N-terminal or C-terminal domains, as well as the lipid environment, appear to restrict the

Three-dimensional BetP Structure to 8 Å by EM

conformational states of BetP under such specific conditions. We therefore conclude that the three distinct protomer conformations in the asymmetric trimer truly represent the physiologically relevant structure of BetP. A similar dependence of transporter conformation on crystal contacts was observed for the multidrug efflux pump AcrB, which also forms either symmetric 30 or asymmetric 27,28 trimers in 3D crystals. The asymmetric form is likewise thought to represent the physiologically relevant state of AcrB, consistent with a transport mechanism involving conformational coupling.31 The different forms of AcrB depend on the space group of the crystal, supporting the premise that crystal contacts may be an important factor also for BetP. The asymmetry in the BetP trimer implies that oligomerization is important for the function of betaine transport. Moreover, it strongly suggests a conformational coupling between the protomers in the membrane. Conformational coupling could simultaneously facilitate the conversion of individual protomers from an outward-facing state into an inward-facing state by reducing the energetic barrier to one or more of the rate-limiting steps in the transport cycle. Higher-resolution data will be required to attain atomically detailed models of the three conformations of BetP and to conclusively assign each protomer to a given conformation in the transport cycle. At the same time, investigations of the function of the BetP monomer are underway in order to determine in what ways the catalytic and regulatory cycles take advantage of the trimeric state.

Materials and Methods Protein expression, purification, and 2D crystallization The plasmid pASK-IBA5betPΔC45 containing an N-terminal StrepII tag was constructed as described previously11 and transformed into E. coli C43 cells.32 Protein expression and membrane preparation were performed as described previously.9 Membranes were solubilized in 1.5% (wt/vol) dodecyl maltoside (Glycon) for 30 min on ice, and the insoluble material was removed by centrifugation (140,000g, 45 min). The supernatant was loaded onto 4 ml of Streptactin resin (IBA GmbH) and washed with 25 ml of 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 10% glycerol, and 0.04% dodecyl maltoside. BetPΔC45 was eluted with 5 mM desthiobiotin. C. glutamicum lipid extracts in 0.15% decylmaltoside were mixed with purified protein at an LPR of 0.15 (wt/wt) and placed into an amini Slide-ALyzer 10K dialysis device (Pierce, Rockford, IL) and dialyzed at 30 °C for 3 weeks against 200–500 ml of dialysis buffer [50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 5% glycerol, 5% 2-methy-2,4-pentanediol, 4 mM CaCl2, and 3 mM NaN3].


Three-dimensional BetP Structure to 8 Å by EM Betaine uptake measurements: Functionality in 2D crystals Uptake of [14C]betaine by BetPΔC45 was measured in E. coli polar lipid proteoliposomes and 2D crystals. Proteoliposomes were prepared in accordance with Schiller et al.8 Two-dimensional crystals were fused with liposomes composed of E. coli polar lipids (Avanti) in the presence of 100 mM potassium Pi (pH 7.5). For this purpose, 2D crystals were mixed at the indicated ratios and extruded 20 times through a polycarbonate filter with a pore size of 400 nm before they were frozen into liquid nitrogen. After gentle thawing, the mixture was incubated for 1 h at 20 °C. The freeze–thaw cycle was repeated three times. The mixture was extruded (15 times) before being used for transport assays. Proteoliposomes and 2D crystals were adjusted to a final concentration of 3–4 μg of BetP with 50 mM Pi (pH 7.5) and 25 mM NaCl containing also 15 μM [14C]betaine (0.1 mCi/ml) and 0.5 μM valinomycin. To establish hyperosmotic conditions, we added proline to the external buffer (0.1– 1.0 Osm/kg). After different time intervals of 5–20 s, samples were filtered rapidly through 0.22-μm GS nitrocellulose filters (Millipore Corp., Germany), and radioactivity was determined by liquid scintillation counting. For the comparison of different LPRs in Fig. 1b, the osmolality of the external buffer was adjusted to 640 mOsm/kg by addition of proline, and uptake was measured at different time intervals of up to 20 s. The measurements were repeated five times. Control experiments with wild-type BetP in proteoliposomes were carried out under the same conditions. Electron crystallography Two-dimensional crystals were stained with 1% uranyl acetate for screening by EM.33 Frozen–hydrated specimens for data collection were prepared by backinjection34 with 10% glucose and transferred into a JEOL 3000SFF electron microscope equipped with a liquid-heliumcooled top-entry stage and a field emission gun operating at 300 kV. Images were taken on Kodak SO-163 electron emulsion films (at a magnification of 45,000× or 53,000×) in spot-scan mode at a total electron dose of 10–20 e/Å2. Image processing Micrographs were selected by optical diffraction, and areas of 6000 × 6000 pixels were digitized on a Zeiss SCAI scanner with a 7-μm pixel size. The Medical Research Council image processing software package35 was used to extract structure factor amplitudes and phases from 72 individual images, after correcting for lattice distortions and the effects of the contrast transfer function. Image data for each tilt angle were merged in-plane with space group p121_b. The tilt geometry and phase origin of each image were refined at 12 Å resolution at the optimal signal-tonoise ratio. Image amplitudes were scaled with an average negative temperature factor B = −300 Å2 to compensate for the resolution-dependent degradation of amplitudes. A 3D map was constructed with the CCP4i package,36 and the map was visualized with the graphics programs Coot and PyMOL.37 NCS-averaged maps were calculated in

Coot and in the CCP4i package using the protomer models (see the text below). Automated rigid-body fitting of X-ray structures to EM maps We carried out an automated rigid-body fitting of the trimeric X-ray structure to the trimeric EM map, as well as of a protomer of the X-ray structure to each of the three protomeric EM maps. The fitting was performed using the CoLoRes tool from the Situs package.14,15 The structure used was the 3.35-Å-resolution X-ray crystal structure of BetPΔN29 (PDB entry 2WIT) with a 45-residue C-terminal truncation, with two missing residues (Asp273 and Pro274) added using Modeller 9v2.38 CoLoRes performed a six-dimensional (three translational degrees of freedom and three rotational degrees of freedom) exhaustive search (accelerated by fast Fourier transform) of the best fit between an experimentally determined map and a map derived from a model atomic structure. The resolution of the derived map was set to 8 Å in the membrane plane and 16 Å normal to the membrane. The grid spacing was 2.3 Å, and the rotational sampling step size was 10°. To enhance the “contour” information in the experimental and calculated maps, we used Laplacian filtering.14 Both L-CC and CC scores are sensitive to the intensity and dimensions of a given map, so absolute values cannot be compared between maps. After ranking by L-CC, the 10 best fits were then locally refined using an off-lattice Powell optimization.39 In the case of protomer 1, the highest-ranking fit of the X-ray protomer to the EM map was shifted by 10.7 Å along the axis normal to the membrane to match its average position in the top seven trimer fits; this fit was then rescored. This adjustment allowed for a better comparison with the trimer fit and is justified given the low resolution of the data along this axis, which is poorest in this particular protomer map. The L-CC scores of the final fits to protomers 1, 2, and 3 were 0.066, 0.125, and 0.072, respectively. Flexible fitting of individual helices to EM maps Starting from the rigid-body protomer fits, the conformation of six TM helix segments was optimized based on the EM density in each of the three protomers, using an automated flexible-fitting approach. These segments are 3c, 3p, 5, 8c, 8p, and 12p. For each segment, 104 putative conformations were generated using the program segsam.40 To generate each conformation, we randomly perturbed all torsion angles in the flexible regions flanking the helix segments, resulting in a rigid-body displacement of the segment. Breaks in the flanking loops were closed using the CCD algorithm. Each conformation was then L-CC rescored along with the rest of the structure using the CoLaCor tool.14 The conformation with the highest L-CC value was then selected, although the top five conformations were typically similar. The highest-ranking conformations for all six segments were then combined with the remainder of the X-ray structure into a complete model for that protomer (two alternate models in the case of protomer 2). To reduce side-chain steric clashes, we energy minimized this model using the steepest-descent algorithm for 1000 steps with the OPLS force field in

380 GROMACS.41 The final L-CC scores were then obtained for each energy-minimized protomer model in its corresponding EM protomer map. The DaliLite program was used for structural alignments.42 Accession numbers

Three-dimensional BetP Structure to 8 Å by EM



The EM density was deposited in the EM data bank with accession code EMD-1756. The flexible-fitting models were deposited in the Protein Model Databank† with accession code PM0076442. 10.

Acknowledgements We thank Winfried Haase for help with the freeze– fracture experiments, Deryck Mills for assistance with electron microscope operation, Janet Vonck and Ingeborg Schmidt-Krey for help with image processing, and Willy Wriggers for assistance with Situs. Caroline Koshy calculated the 8-Å maps of Mhp1. Susanne Morbach and Vera Ott contributed valuable discussions to the BetP transport mechanism. This work was supported by the International Max Planck Research School Frankfurt.

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