J. Mol. Biol. (1992) 223, 1155-1165
Two-dimensional Crystals of Escherichia coli Maltoporin and Their Interaction with the Maltose-binding Protein Kathrin A. Statiert Department of Microbiology, Biocentre University of Basel, CH-4056 Basel, Switzerland
Andreas Hoenger and Andreas EngelS M. E. Miiller-Institute
for High-Resolution Electron Microsqy University of Base1 CH-4056 Basel, Switzerland
at the B&en&e,
(Received 16 May 1991; accepted 16 October 1991) We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stamed membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm))‘. Furthermore, we have coreconstituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with One of the hexagonal arrays was found to bind maltose-binding protein maltoporin. molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.
Keywords: maltoporin;
maltose-binding protein; 2-D membrane protein crystals; image processing; multivariate statistical analysis
1. Introduction Maltose transport into Escherichia wli is accomplished by a sophisticated system of proteins located in the outer membrane, the periplasmic space, the inner membrane and the cytoplasm. The system has been studied extensively as a model for transport involving the co-ordinated action of several proteins that are located in different cellular compartments (Schwartz, 1987; Hengge & Boos, 1983). t Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, U.K. $ Author to whom all correspondence should be addressed at: M.E. Miiller-Institute for High-Resolution Electron Microscopy at the Biocentre, University of Basel, Klingelbergstrasse 70, CH-4056 B-1, Switzerland. 0922-2836/92/041155-11
)03.99/o
The first step in maltose uptake, the diffusion of maltose across the outer membrane, is facilitated by an integral protein of the outer membrane called maltoporin. This protein, encoded by the gene ZamB,was first characterized through its function as phage L receptor (Randall-Hazelbauer & Schwartz, 1973). It has a relative molecular mass of 47,999 and exhibits some distinct similarities with unspecific porins in secondary, tertiary and quaternary but not primary structure (Clement & Hofnung, 1981). Thus, the protein contains predominantly B-pleated sheets and few if any a-helical regions, and assumes an extremely stable trimeric configuration in satu. A low-resolution three-dimensional (3-w) model obtained from projections of negatively stained twoQ Abbreviations used: 3-D, three-dimensional; 2-D, two-dimensional; MBP, maltose-binding protein; octyl-POE, octyl polyoxyethylene; LPR, lipid-to-protein ratio (w/w). 1155
0 1992 Academic Press Limited
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Figure 1. Negatively stained maltoporin arrays reconstituted with E. coli lipids. (a) and (b) at LPR = @l, tubes and vesicles coexist; while (c) at LPR = 1.0, multilayered sheets assemble during detergent removal. (al) Vesicles are flattened during negative staining and the rotational misalignment, of diffraction spots indicates that top and bottom layers are not in register. The spots of 1 layer are arranged in a hexagonal pattern typical for trigonal lattices assembled
Maltoporin
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dimensional (2-D) maltoporin crystals (Lepault et al., 1988) reveals stain-filled channel triplets that appear to merge into a single outlet, a configuration that is similar to that of other porin channels (Engel et al., 1985; Chalcroft et al., 1987). Functionally, maltoporin differs considerably from other porins: in addition to providing for unspecific diffusion of solutes up to a molecular mass of 606 Da, the passage of maltodextrins up to at least twice that size is specifically mediated by maltoporin channels. The single channel conductance is four times lower than that of OmpF porin and is specifically inhibited by maltodextrins (Dargent et al., 1987). Moreover, maltoporin channels fluctuate with a unit step size, whereas in the case of OmpF, the closing step amounts to about one-third of the opening step. The maltodextrin-binding properties of maltoporin have been studied in some detail (Ferenci & Boos, 1980). Affinity measurements show that the binding of maltose itself is quite weak, with a dissociation constant in the millimolar range. Increasing chain length of linear maltodextrins drastically increases their binding affinity for maltoporin; thus, maltohexaose binds with a micromolar dissociation constant. The transport of maltose across the outer membrane is not an active process but proceeds strictly down a concentration gradient. There is, however, a scavenger protein in the periplasm that keeps the concentration of free maltose at micromolar levels and thus maintains net maltose influx. This protein is called the maltose-binding protein (MBP) and is encoded by the gene m&E. Its relative molecular mass is 40,099, and it exhibits tight binding of maltose and maltodextrins with similar affinities, all in the micromolar range (Szmelcman et al., 1976). It is known to interact with the MalFMalG-MalK complex that constitutes the active maltose transport system in the inner membrane (Shuman, 1982). On the basis of genetic evidence (Bavoil et al., 1983), it has always been assumed that MBP also binds to maltoporin, picking up a substrate molecule as it emerges from the pore, and subsequently dissociates from maltoporin to shuttle the substrate to the inner membrane (Wandersman et al., 1979). This model was given further support by a reported influence of MBP on maltoporin channel activity (Neuhaus et al., 1983). However, clear biochemical demonstration of such an interaction has
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proven difficult. Although immobilized MBP will bind maltoporin, this binding is not reversible even by high concentrations of maltose as would be expected (Bavoil & Nikaido, 1981). Analytical ultracentrifugation of maltoporin-MBP mixtures shows two bands corresponding to maltoporin trimer and MBP monomer, and fails to reveal the presence of a stable maltoporin-MBP complex (A. Lustig, K. A. Stauffer dz A. Engel, unpublished results). Furthermore, the stabilization of maltoporin conductance by MBP has not been reproduced (Dargent et al., 1987). Here, we report on MBP-maltoporin interactions studied by electron microscopy and image processing of 2-D crystals reconstituted from lipids, MBP and maltoporin. Substoichiometric binding is suggested by SDS/polyacrylamide gel electrophoresis of such arrays after extensive washing. A difference map from averaged unit cells of MBPmaltoporin arrays and an MBP-free control displays three symmetry-related MBP binding sites per maltoporin trimer. As assessed by multivariate statistical analysis, not more than one of these is likely to be occupied at a given time. The methods used here could be of general interest for the study of the structure of a soluble protein and its binding to an insoluble ligand reconstituted into a 2-D regular array. 2. Materials
and Methods
E. co5 W3110 were grown in M9 medium supplemented with 615% (w/v) Casamino acids and 92% (w/v) maltose.
Maltose-binding protein was purified from those cells according to the method of Ferenci Lb Klotz (1978). In some experiments, the maltose was eliminated by dialysis against 2 M-guanidium . HCl in 20 mna-Hepes (pH 7.4) against Hepes buffer alone. followed by dialysis Maltoporin was extracted from cells broken by passage through a French pressure cell and subsequently purified by a combination of anion-exchange chromatography, chromatofocussing and gel filtration as described (Stauffer et al., 1990). The final product migrated as a single band on SDS/polyacrylamide gel electrophoresis. No residual lipopolysaccharide could be detected by silver staining. OmpF porin was extracted from E. co& BE cells (strain BZB 3333) and isolated ss described (Holzenburg et al., 1989). Phospholipid (phosphatidyl ethanolamine, Sigma type V, from E. CA) was dried in a stream of nitrogen at 37°C and solubilized to either 1 or 10 mg/ml in buffer
from trimeric complexes and reveal unit cell dimensions of a = b = 7.9 nm. (a2) The correlation average, however, exhibits an apparent g-fold symmetry. This is due to 2 possible angular orientations around the 3-fold symmetry axis of the maltoporin trimers that differ by n. (a3) and (a4) The 2 correlation averages, which were calculated from 1 of the 2 layers using 2 different references, illustrate this point. (b) Rarely, tubes open up to grow out as single sheets that reveal a rectangular crystal habit as suggested by (bl) the diffraction pattern and (b2) the averaged projection. One unit cell contains 2 maltoporin trimers in opposite angular orientation and exhibits dimensions of a = 7.9 nm, b = 137 nm. Maltoporin monomers show a major and a minor domain separated by a groove. (c) The hexagonal pattern is distinctly visible on the micrograph of a double-layered maltoporin sheet (unit cell dimensions a = b = 94 nm). (cl) The optical diffraction pattern is quite different from that of vesicles, exhibiting strong (1,l) orders and no rotational misalignment. (~2) The averaged projection also reveals a different unit cell morphology from the other crystal forms, but it can be explained by a counter clockwise rotation of the maltoporin trimer by 12”. The scale bars on the micrographs represent 200 nm in (a) and (b), 100 nm in (c) and (20 nm)-’ in the diffraction patterns.
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Figure 2. Reconstituted maltoporin-MBP arrays. (a) and (b) As without MBP, vesicles and tubes are found at LPR = O-1, while (c) multilayered sheets are formed at LPR = 1.0. (a) MBPs are randomly adsorbed to the free carbon film, whereas they appear to be partially ordered along the lattice lines of the single-layer maltoporin membrane. (al) The diffraction pattern indicates some distinct (2,2) orders, which correspond to a spatial frequency of approximately (2 nm)-‘. (a2) The average projection resembles that of the hexagonal maltoporin lattice without MBP (see Fig. l(a2)) and possesses an apparent 6-fold symmetry as well. (b) No ordered MBP decoration is discernible on maltoporin tubes, although randomly dispersed MBP is visible in the background. (bl) The diffraction pattern shows the top and bottom layer of the collapsed tube built from a rectangular maltoporin lattice. (b2) The average from 1 layer reveals 2 trimers per rectangular unit cell, similar to the average from the rectangular layer shown in Fig. l(b2). (c) Randomly arranged particles can be seen on the multilayered sheet reconstituted at LPR = 1.0. (cl) The diffraction pattern and (~2) the average closely match those from the MBP-free control. The scale bars on the micrographs represent 200 nm in (a) and (b), 100 nm in (c) and (2.0 nm)-’ on the diffraction patterns.
Maltopwin
Maltose-binding
Figure 3. Samples of coreconstituted MBP and maltoporin analysed on SDS/polyacrylamide gel electrophoresis. Lane a, membranes after reconstitution; lanes b to e, supernatants of 4 consecutive washes with MBP-free buffer; lane f, maltoporin-MBP membranes at the end of this washing procedure.
containing 7.5% octyl polyoxyethylene (octyl-POE, a kind gift from J. P. Rosenbusch). In a typical reconstitution experiment, 40 pl of maltoporin (10 mg/ml in buffer containing 1 y. octyl-POE) wss mixed with 40 ~1 of phospholipid solution, 280 ~1 of Hepes buffer (20 mM-Hepes (pH 7-O), 10 mna-M&Y,, lOO.mNaCl, 62 mr+r-EDTA, 3 mnr-NaN,, 02 mM-dithiothreitol) and 40 ~1 of MBP (10 mgJm1). The final maltoporin concentration was 1 mg/ml, and the molar ratio of MBP to maltoporin was slightly greater than 1. The ratio of phospholipid to maltoporin was either 61 or 1 (w/w). After passing the sample through a 645 pm Millipore filter it w&8 dialysed against a total of approximately 21 of Hepes buffer for 2 days in a continuous-flow apparatus as described (Engel et al., 1988). During the first 36 h the temperature was held constant at 37°C then it was decreased to 22°C in a linear ramp over 12 h. The association of MBP with different porin arrays was sssayed by SDS/polyacrylamide gel electrophoresis. A volume of 406 ~1 containing either O-4 mg of OmpF porin or O-4 mg of maltoporin coreconstituted with 04 mg of MBP in the presence of phospholipids was centrifuged in a Heraeus Biofuge A at 5000 revs/min for 5 min, the supernatant saved, and the pellet resuspended in 400~1 of Hepes buffer. This procedure ws.s repeated 4 times, but the pellet was resuspended in 40 ~1 of buffer after the last wash. A portion (10 ~1) of the initial solution, of each supernatant and of the resuspended pellet were applied to an SDS/l@5Oh polyacrylamide gel. Reconstituted protein-lipid membranes were adsorbed to electron microscope grids covered by carbon-topped formvar films that were rendered hydrophilic by glow discharge at low pressure in air. After washing in Tris buffer (10 mm-Tris. HCl, pH 7) the membranes were negatively stained with 075% (w/v) many1 formate (pH 42) ammonium molybdate (pH 66). or 2% (w/v) Alternatively, adsorbed membranes were washed in double-distilled water, frozen in liquid nitrogen, freezedried in a Balzer BAF 300 unit at -80°C and unidirectionally shadowed with tantalum/tungsten at an elevation angle of 45” at the same temperature. Images were recorded on Kodak SO-163 lilm at a nominal magnification of 50,000 x and a dose of approximately 2000 e/rim’ using a Hitachi H-7060 transmission
Protein
Interaction
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electron microscope operated at 100 kV. The magnification was calibrated using negatively stained oat&se crystals. Micrographs were evaluated for focus, astigmatism and drift as well as for crystallinity of reconstituted membranes by optical difliaction (see Aebi et oL, 1973). Suitable areas were digitized using an Eikonix 850 CCD camera equipped with a Zeiss S-planar objective lens. To eliminate noise introduced by gain and bias variations of the CCD-array, an empty frame was recorded and stored for on-line normalization. In addition, 3 x 3 pixel areas were averaged and stored as single pixels separated by @6 nm. Several methods were applied for calculating averaged projections of maltoporin unit cells with and without MBP, mainly taking advantage of the SEMPER image processing system (Saxton et aE., 1979). First, straightforward Fourier peak llltration (see Aebi et al., 1973) was used for a rapid evaluation of unit cell morphologies and crystal quality. Second, correlation averaging (Sexton & Baumeister, 1982) allowed the elimination of residual lattice disorder by locating the unit cells precisely using a reference that included 3 to 4 maltoporin trimers. In addition, correlation functions were used to discriminate 2 angular orientations of the maltoporin trimers with respect to the 3-fold axis. To increase the sensitivity, references were filtered by the clipped power spectrum of the difference map calculated from the 2 possible angular orientations. Third, subframes containing 4 trimers were extracted at the position of cross-correlation peaks and submitted to a multivariate statistical classification exercising a program package kindly provided by J. P. Bretaudiere (see Frank at al., 1988). The classification algorithm used “dynamic clouds” clustering for partitioning the images into 49 clusters and rejecting the images that could not be assigned to any of the 49 clusters. The clusters were then further partitioned by hierarchical sacending classification, yielding a clessification tree. Clusters or classes of MBP-decorated maltoporin trimers determined in this way were averaged and the difference images of such averages and maltoporin controls calculated. The weight of the control was adapted to minimize the dynamic power of the difference map. Surface reliefs were calculated from averages of freeze-dried, metal-shadowed maltoporin-membranes aa described (Smith & Kistler, 1977).
3. Results In agreement with a previous report (Lepault et al., 1988), maltoporin could be reconstituted into vesicles and tubes with E. coli phospholipids at a lipid-to-protein ratio (w/w; LPR) of 91 (Fig. l(a) and (b)). The packing arrangement of the maltoporin trimers is hexagonal in vesicles and rectangular in tubes. Top and bottom layers of collapsed vesicles were usually not in register, as visible from the diffraction pattern (Fig. l(a1)). The Fourier peak filtration of one layer locally revealed a p3 lattice (a = b = 79 nm) with significant irregularities (not shown), but a straightforward correlation average over a large area exhibited a rather puzzling p6 symmetry (Fig. l(a2)). A closer analysis, however, showed that maltoporin trimers were inserted in two distinct angular orientations that differed by n (Fig. l(a3) and (a4)). Such in-plane phase transitions involving rotation of trimers
K. A. Stauffer et al.
maltoporin arrays with a,& Figure 4. Correlation averages and multivariate statistical classification of different multiva without MBP. (a) (left) A 3-fold symmetry is evident in the average of 900 angularly oriented subframes containing 4 unit cells extracted from a single layer of a collapsed vesicle. The average calculated from 2500 MBP-decorated subframes is shown in (a) on the right side. (a) (centre) The difference map of these averages indicates the presence of additional mass in 3-fold related positions (1 to 3), the most likely binding sites of MBP. (b) Structural variations are characteristic for negatively stained aIT8yS. They are pronounced in collapsed vesicles as illustrated by averages of images not contained in clusters, images of the two major clusters, and images of the 3 major classes (from left to right). (c) Prominent particles become discernible on cluster-averages of MBP-decorated vesicles. They are localized at position 2 (1st row) or position 3 (2nd row) in the cluster-averages shown. (d) Difference maps calculated by subtrttction of the weighted control from individual cluster-averages were averaged after translational alignment (position 2: left; position 3: centre). Contours of the MBP projection delineating the edge and the core of the molecule, respectively, are drawn over the MBP-free maltoporin unit cell (right). The scale bars represent 5 nm.
Maltoporin
Maltose-binding
around their S-fold axis have been reported for OmpF porin (Dorset et al., 1989). Occasionally, tubes initiated the crystallization of sheets that revealed the large, single-layered rectangular unit cell (a = 7.9 nm, b = 13.7 nm) containing two maltoporin trimers (Fig. l(b)) with particular clarity. The projection of the rectangular unit cell displayed major and minor domains of the maltoporin monomer that were separated by a distinct groove. In general, the crystal order of negatively stained tubes and single layers (Fig. l(b)) was better than that of vesicles (Fig. l(a)), which were severely distorted as a result of flattening during negative staining/air drying. At LPR = 1, a new crystal habit was found. These large, double-layered hexagonal sheets (Fig. l(c)) with unit cell size a = b = 8.4 nm were well ordered as indicated by sharp diffraction spots extending to (2nm)-’ (Fig. l(c1)). The lack of rotational misalignment in these diffraction patterns suggested that the layers stacked in register, indicating a strong interaction between maltoporin trimers of different layers. Averaged projections showed trimeric pores that resembled those of the rectangular and hexagonal lattices (Fig. l(a3) and (b2)), but that were rotated by 24” about the central 3-fold axis of symmetry (Fig. l(c2)). This rotation was paralleled by a 13% increase in the unit cell area, thus providing space to accommodate additional lipids. All three crystal forms could be reconstituted in the presence of an excess of MBP, and for all three forms well-ordered lattices were observed (Fig. 2). A non-random position and orientation of membraneattached MBP was suggested by rows of particles along the direction of lattice lines on disrupted maltoporin vesicles (Fig. 2(a)). This is most distinct when the micrograph is viewed both at a glancing angle and along a lattice line. However, only a close comparison of the average in Figure 2(a2) with that in Figure l(a2) indicated a subtle reduction of the gap between subunits of adjacent pores, which could be the result of some additional mass. On tubes (Fig. 2(b)) and sheets (Fig. 2(c)), MBP particles were not deposited in an ordered fashion. Repeated washing of membranes reconstituted in the presence of MBP with equal buffer volumes revealed that MBP was membrane-bound, but that less than one MBP molecule per maltoporin trimer remained attached as suggested by the relative intensities of bands on SDS/polyacrylamide gel electrophoresis (Fig. 3). Controls with arrays from OmpF porin and dimyristoyl phosphatidyl choline incubated with MBP showed no binding. This very simple binding assay confirms that binding of MBP to maltoporin, under the conditions used in our reconstitution experiments at about 25 j.lM-protein, occurs in less than stoichiometric amounts. As illustrated by Figure l(a), maltoporin trimers were inserted in two distinct angular orientations into hexagonal vesicles. Therefore, individual pores, localized by their cross-correlation signal with a suitable reference (i.e. average in Fig. l(a2)), were
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extracted and their orientation determined by comparison with two references of opposite orientation (Fig. l(a3, a4)). Approximately half of the unit cells had to be rotated by 7~for subsequent averaging. The average of 900 maltoporin trimers generated in this way (Fig. 4(a), left) exhibited a stainpenetrated triangular pore similar to that of rectangular-tubes (Figs l(b) and 2(b)). The major and minor domains of maltoporin monomers were distinct and allowed the hand of the pore to be determined. The two maltoporin domains-were also visible on the average of 2590 maltoporin trimers from MBP-decorated disrupted vesicles (Fig. 4(a), right), but they appeared to be slightly larger than those of MBP-free vesicles. The difference map presented in Figure 4(a) (centre) indicated three prominent binding sites of MBP per trimer, which were located between adjacent pores. To separate MBP-free unit cells from those carrying one or several MBP molecules, oriented projections comprising four maltoporin trimers from decorated vesicles (Fig. 2(a)), tubes (Fig. 2(b)) and sheets (Fig. 2(c)) were submitted to multivariate statistical analysis (van Heel & Frank, 1981; van Heel, 1984; Frank et al., 1988). Bretaudiere’s classification algorithm (Frank et al., 1988) was used to determine clusters of similar images in a first step and to subsequently assess the similarity of different clusters by a hierarchical ascending classification scheme. This classification tree allowed clusters to be partitioned into classes of ascending order for subsequent averaging. Because this approach has been critically debated by van Heel (1989), we have tested its sensitivity and reproducibility with our own data. First, MBP-free maltoporin trimers were analysed before angular orientation. The algorithm divided unit cells into two major classes, the “up” and “down” orientations. In addition, we found that the ratio of up and down trimers was in excellent agreement with the result from correlation measurements. Second, refinement runs were done on various data sets, omitting images that could not be assigned to prominent clusters, thereby yielding a more pronounced clustering of the remaining images. The fact that we found only minor changes between averages calculated from images of a particular cluster during several refinement steps indicated appropriate functioning of the classification scheme (data not shown). As a next step, we analysed the variability of negatively stained, angularly aligned, MBP-free maltoporin trimers from single-layered arrays. As illustrated by Figure 4(b) (left), the average of projections comprising four pores that did not belong to any identified cluster (28.8% of the 900 cells analysed) exhibited a strong similarity to the undecorated pore calculated by correlation averaging (Figs l(a), right and 4(a), left). Variations due to stain irregularities and structural damage during dehydration were demonstrated by the averages of the two major clusters containing 47 y0 and 4.3 y0 of the cells, respectively, and the averages of the three
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Figure 5. Freeze-dried, metal-shadowed maltoporin (a) tubes and (b) sheets show regular fine structures to a resolution of 2.5 nm (see (al)). Although reconstituted in the presence of MBP, no random or regular decoration of such surfaces occurs, whereas MBP molecules appear to adhere to the carbon film. (a2) and (a3) The surface relief reconstructed from the 2 tubes exhibit 2 trimeric depressions per rectangular unit cell, whereas (bl) and (b2) trimeric depressions are arranged in a hexagonal lattice at the surface of maltoporin sheets. The scale bars represent 50 nm in (a), 100 nm in (b) and (3.0 nm)-’ in (al).
Maltoporin
Maltose-binding
major classes (192%, 291% and 31.9%; Fig. 4(b), from left to right). In contrast to this, MBP-decorated maltoporin trimers in disrupted vesicles revealed outstanding features that suggested systematic binding of MBP. The first row in Figure 4(c) shows cluster-averages from a classification of 2500 areas containing four maltoporin trimers with one MBP bound at position 2 (see Fig. 4(a), centre), and the second row shows MBP in position 3. Two MBP molecules were bound in the vicinity of one pore quite frequently, but they consistently occupied sites of different maltoporin trimers (155%, data not shown), while less than 1 O/Oshowed more than two additional protuberances. In all, 296% of the 2500 areas analysed were not assigned to any clusters. Their average was indistinguishable from that of the MBP-free control, suggesting that about 30% of the unit cells were MBP-free. Since the shape of the bound MBP was obscured by the underlying maltoporin array, difference maps of all cluster-averages and appropriately weighted projections of MBP-free maltoporin trimers were calculated. Correlation functions between these difference maps and those showing the MBP in positions 1, 2 and 3, respectively (Fig. 4(a), centre) were used to refine the alignment of bound MBP for subsequent averaging. Maps exhibiting a correlation coefficient above 66 were then merged to calculate the MBP average for position 1, position 2 (Fig. 4(d), left), and position 3 (Fig. 4(d), centre). These averages were angularly aligned to produce the final MBP map containing 42% of the unit cells carrying at least one MBP. The contour of the MBP molecule was superimposed on the maltoporin trimer projection to illustrate the position of the MBP on the rim of the pore (Fig. 4(d), right). To further elucidate the interaction between MBP and maltoporin integrated in tubes and sheets, such membranes were freeze-dried and metal-shadowed (Fig. 5). The surfaces of both maltoporin tubes and sheets appeared to be very clean, with no traces of randomly or specifically bound MBP. Structural preservation to 25 nm (Fig. 5(al)) allowed the surface reliefs of both crystal habits to be reconstructed and the entrances of the pores to be visualized. As displayed in Figure 5(a2) and (a3), the tubes exhibited two triplet openings per rectangular unit cell, one of them rotated by n with respect to the other. Triplet openings arranged on a hexagonal lattice were clearly discernible on the surface of the sheets as well (Fig. 5(bl) and (b2)). Vesicles, on the other hand, were not well ordered enough for such an analysis. 4. Discussion The results presented here document binding of E. coli MalE protein, the maltose-binding scavenger located in the periplasmic space, to LamB protein, the maltoporin in the outer membrane. So far, no biochemical data are available that would allow this interaction to be characterized quantitatively,
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although conceptually, MBP-maltoporin binding is appealing, considering the functional aspects of the maltose transport system. Crosslinking experiments performed to show the existence of a MBPmaltoporin complex in solution (data not shown) have failed in our hands, and suggest that the presence of detergent, or the absence of lipid, may interfere with complex formation. We cannot assess at this point which of the two proteins is affected; it may be noted, however, that the binding of solubilized maltoporin to phage 1 seems to be somewhat impaired as well (Randall-Hazelbauer & Schwartz, 1973). With maltoporin reconstituted into a lipid bilayer, the MBP-maltoporin interaction becomes rather less intractable. The availability of wellordered reconstituted maltoporin membranes has tempted us to study the complex structurally, combining electron microscopy with digital imageprocessing. Indeed, we have been able to observe of maltoporin arrays with MBP, decoration although a majority of the possible MBP-binding sites appear to be unoccupied while some excess MBP remains at random positions. Fully induced bacteria are estimated to synthesize about 2 x lo4 molecules of MBP and up to 1 x lo5 molecules of maltoporin per cell (Schwartz, 1987). This corresponds to concentrations of around 50 PM over the whole cell. Moreover, since the distribution of both proteins is strictly limited to the periplasmic space and the outer membrane, respectively, local concentrations could be expected to reach several hundred PM. It is not surprising, therefore, that the interaction between the two proteins seems to require only a moderate affinity constant. On the other hand, when MBP binds a maltodextrin molecule, even a small conformational change in the maltoporin-binding domain of MBP can be expected to lead to dissociation of the complex if the binding affinity is not very high. MBP molecules properly bound to maltoporin arrays could be identified by correlation averaging and multivariate statistical analysis. Multivariate statistical classification (van Heel, 1989) separates images into subclasses according to a straightforward similarity criterion: it is indicated by the clustering of vectors in an N-dimensional space (where N = n x m) representing images containing n x m pixels. After extensive testing we have used Bretaudiere’s classification algorithm (Frank et al., 1988) to partition images of maltoporin trimers with and without MBP. This algorithm first determines the clustering of images by the dynamic cloud technique and then partitions the clusters by a hierarchical ascending classification scheme. The respective cluster-averages and class-averages of MBP-decorated maltoporin trimers were then further analysed for their angular orientation with respect to one another, and compared to averages from MBP-free controls. For single-layered hexagonal maltoporin lattices, this approach reproducibly yielded a distinct indication of three possible MBP-binding sites per malto-
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porin trimer, of which not more than one appears to be occupied in most cases. No binding site could be identified on maltoporin tubes or double-layered sheets. Further investigation of tubes and double-layered sheets by freeze-drying and metal-shadowing produced an explanation of this finding: the surface reconstructions in Figure 5 show clearly that, at least in our hands, both tubes and double-layered sheets expose trimeric openings at their surfaces. In analogy to an assignment made for OmpF porin (Engel et al., 1985; Hoenger et al., 1990), we conclude this surface to be the extracellular face of maltoporin (as opposed to the assignment made by Lepault et al., 1988). MBP would therefore have to bind on the inner surface of narrow tubes, or between the layers of tightly interacting double sheets, a very unfavourable process in each case. The complete lack of MBP decoration on the outer surfaces of tubes would seem to support our conclusion about their sidedness. After aligning and averaging the most prominent cluster-averages containing 42% of the unit cells decorated with at least one molecule of MBP, the rough outline of the bound MBP becomes visible. The projection shows a triangular-shaped molecule with dimensions of approximately 5 x 4 nm. The X-ray structure (Spurlino et al., 1991) establishes MBP to be a triangular-shaped disk with dimensions of about 6 nm x 4 nm x 3 nm. Considering the similarity in the overall shape of the molecule, we propose that our map shows MBP in a projection with its shortest axis perpendicular to the plane of the membrane. Located for the greater part on the rim of the pore, the MBP molecule appears to project out over the pore and thus to occupy just the kind of position one would expect to be optimal for its interaction with incoming maltodextrins. It will be interesting to map the protein-protein interface on the X-ray structures of both MBP and, once it becomes available, maltoporin. The structural basis of interactions between integral membrane proteins and soluble proteins has not been studied in great detail so far, and the MBP-maltoporin pair might serve as a model system for other processes that involve the association and dissociation of a soluble protein from a membrane surface. We thank Jiirg Rosenbusch and Malcolm Page for stimulating discussions, Ueli Aebi and John Berriman for critical comments on the manuscript, and Andreas Hefti, Ariel Lustig and Ariane Hardmeyer for technical assistance. We are grateful to Jean-Pierre Bretaudiere for the programs for multivariate statistical analysis and classification. The work was supported by Swiss Kational Science Foundation grants 31-25684.88 to A.E., and 3.29485 to J. P. Rosenbusch, and the Maurice E. MiillerFoundation of Switzerland.
References Aebi,
U., Smith, P. R., Dubochet, J., Henry, C. & Kellenberger, E. (1973). A study of the structure of the T-layer of Bacillus brevis. J. Supramol. Struct. 1,
498-522.
et al. Bavoil, P. & Nikaido, H. (1981). Physical interaction between the phage I receptor protein and the carrierimmobilized maltose-binding protein of E. co& J. Biol. Chem. 256, 11385-11388. Bavoil, P., Wandersman, C., Schwartz, M. & Nikaido, H. (1983). A mutant form of maltose-binding protein of E. coli deficient in its interaction with bacteriophage 1 receptor protein. J. Bucteriol. 155, 919-921. Chalcroft, J. P., Engelhart, H. t Baumeister, W. (1987). Structure of the porin from a bacterial stalk. FEBS Letters,
211, 53-58.
Clement, J. M. & Hofnung, M. (1981). Gene sequence of the 1 receptor, an outer membrane protein of E. coli K12. Cell, 27, 507-514. Dargent, B., Rosenbusch, J. bz Pattus, F. (1987). Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E. coli outer membrane. FEBS Letters, 220, 136-142. Dorset, D. L., Massalski, A. K. & Rosenbusch, ?J. P. (1989). In-plane phase transition of an integral membrane protein: nucleation of the OmpF matrix porin rectangular polymorph. Proc. Nat. Acad. Sci., U.S.A. 86, 6143-6147. Engel, A., Massalski, A., Schindler, H., Dorset, D. L. & Rosenbusch, J. P. (1985). Porin channel triplets merge into single outlets in E. coli outer membranes. Nature
(London),
317, 643-645.
Engel, A., Holzenburg, A., Stauffer, K., Rosenbusch, J. & Aebi, U. (1988). A novel reconstitution method for inducing the formation of regular 2-dimensional arrays of membrane proteins and lipids. Proc. 46th Ann. Meeting Electron Microsc. Sot. Amer. (Bailey. G. W., ed.), pp. 152-153, San Francisco Press, San Francisco, CA. Ferenci, T. & Boos, W. (1980). The role of E. coli 1 receptor in the transport of maltose and maltodextrins. J. Supramol. Struct. 13, 101-116. Ferenci, T. & Klotz, U. (1978). Affinity chromatographic isolation of the periplasmic maltose binding protein of E. coli. FEBS Letters, 94, 213-217. Frank, J., Bretaudiere, J. P., Carazo, J. M., Verschoor, A. & Wagenknecht, T. (1988). Classification of images of biomolecular assemblies: a study of ribosomal subunits of E. coli. J. Microsc. 150, 99-115. Hengge, R. C Boos, W. (1983). Maltose and lactose transport in E. coli. Examples of two different types of concentrative transport systems. Biochim. Biophys. Acta, 737, 443-478.
Hoenger, A., Gross, H., Aebi, U. & Engel, A. (1996). Localization of the lipopolysaccharides in metalmembranes. shadowed reconstituted lipid-porin J. Struct. Biol. 103, 185-195. Holzenburg, A., Engel, A., Kessler, R., Manz, B. J., Lustig, A. & Aebi, U. (1989). Rapid isolation of OmpF porin-LPS complexes suitable for structurefunction studies. Biochemistry, 28, 4187-4193. Lepault, J., Dargent, B., Tichelaar, W., Rosenbusch, J. P., Leonard, K. & Pattus, F. (1988). Threedimensional reconstruction of maltoporin from electron microscopy and image processing. EMBO J. 7, 261-268. Keuhaus, J.-M., Schindler, H. & Rosenbusch, J. P. (1983). The periplasmic maltose-binding protein modifies the characteristics of maltoporin. channel-forming EMBO J. 2, 1987-1991. Randall-Hazelbauer, L. & Schwartz, M. (1973). Isolation of the bacteriophage lambda receptor from E. coli. J. Bacterial.
116, 1436-1446.
Saxton, W. 0. t Baumeister,
W. (1982). The correlation
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Maltose-binding
averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127-138. Saxton. W. O., Pitt, T. J. & Horner, W. (1979). Digital image processing: the SEMPER system.
Ultramicroscopy,
4, 343-354.
Schwartz, M. (1987). The maltose regulon. In Escherichia typhimurium (Neidhardt, F. C., coli and Salmonella ed.), vol. 2. pp. 148221502, American Society for Microbiology, Washington, DC. Shuman, H. A. (1982). Active transport of maltose in E. coli K12. J. Biol. Chem. 257, 54555461. Smith, P. R. & Kistler, J. (1977). Surface reliefs computed from micrographs of heavy metal-shadowed specimens J. Ultrastruct. Res. 61, 124-133. Spurlino. J. C., Lu, G.-Y. & Quiocho, F. A. (1991). The 23-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J. Biol. Chem,. 266. 520225219.
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Stauffer, K. A., Page, M. G. P., Hardmeyer, A., Keller, T. A. & Pauptit, R. A. (1990). Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli. J. Mol. Biol. 211, 297-299. Szmelcman, S., Schwartz, M., Silhavy. T. J. & Boos, W. (1976). Maltose transport in E. coli K12. Eur. J. Biochem. 65, 13-19. van Heel, M. (1984). Multivariate statistical classification of noisy images (randomly oriented biological macromolecules). Ultramicroscopy, 13, 165-184. van Heel, M. (1989). Classification of very large electron microscopical image data sets. Opt&, 82, 114-126. van Heel, M. & Frank, J. (1981). Use of multivariate statistics in analyzing the images of biological macromolecules. Ultramicroscopy, 6, 187-194. Wandersman, C., Schwartz, M. & Ferenci, T. (1979). E. coli mutants impaired in maltodextrin transport.
.J.
by R. Huber
Bacterial.
140,
l-13.
Two-dimensional Crystals of Escherichia coli Maltoporin and Their Interaction with the Maltose-binding Protein Kathrin A. Statiert Department of Microbiology, Biocentre University of Basel, CH-4056 Basel, Switzerland
Andreas Hoenger and Andreas EngelS M. E. Miiller-Institute
for High-Resolution Electron Microsqy University of Base1 CH-4056 Basel, Switzerland
at the B&en&e,
(Received 16 May 1991; accepted 16 October 1991) We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stamed membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm))‘. Furthermore, we have coreconstituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with One of the hexagonal arrays was found to bind maltose-binding protein maltoporin. molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.
Keywords: maltoporin;
maltose-binding protein; 2-D membrane protein crystals; image processing; multivariate statistical analysis
1. Introduction Maltose transport into Escherichia wli is accomplished by a sophisticated system of proteins located in the outer membrane, the periplasmic space, the inner membrane and the cytoplasm. The system has been studied extensively as a model for transport involving the co-ordinated action of several proteins that are located in different cellular compartments (Schwartz, 1987; Hengge & Boos, 1983). t Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, U.K. $ Author to whom all correspondence should be addressed at: M.E. Miiller-Institute for High-Resolution Electron Microscopy at the Biocentre, University of Basel, Klingelbergstrasse 70, CH-4056 B-1, Switzerland. 0922-2836/92/041155-11
)03.99/o
The first step in maltose uptake, the diffusion of maltose across the outer membrane, is facilitated by an integral protein of the outer membrane called maltoporin. This protein, encoded by the gene ZamB,was first characterized through its function as phage L receptor (Randall-Hazelbauer & Schwartz, 1973). It has a relative molecular mass of 47,999 and exhibits some distinct similarities with unspecific porins in secondary, tertiary and quaternary but not primary structure (Clement & Hofnung, 1981). Thus, the protein contains predominantly B-pleated sheets and few if any a-helical regions, and assumes an extremely stable trimeric configuration in satu. A low-resolution three-dimensional (3-w) model obtained from projections of negatively stained twoQ Abbreviations used: 3-D, three-dimensional; 2-D, two-dimensional; MBP, maltose-binding protein; octyl-POE, octyl polyoxyethylene; LPR, lipid-to-protein ratio (w/w). 1155
0 1992 Academic Press Limited
1156
K. A. Stauffer et al.
Figure 1. Negatively stained maltoporin arrays reconstituted with E. coli lipids. (a) and (b) at LPR = @l, tubes and vesicles coexist; while (c) at LPR = 1.0, multilayered sheets assemble during detergent removal. (al) Vesicles are flattened during negative staining and the rotational misalignment, of diffraction spots indicates that top and bottom layers are not in register. The spots of 1 layer are arranged in a hexagonal pattern typical for trigonal lattices assembled
Maltoporin
Maltose-binding
dimensional (2-D) maltoporin crystals (Lepault et al., 1988) reveals stain-filled channel triplets that appear to merge into a single outlet, a configuration that is similar to that of other porin channels (Engel et al., 1985; Chalcroft et al., 1987). Functionally, maltoporin differs considerably from other porins: in addition to providing for unspecific diffusion of solutes up to a molecular mass of 606 Da, the passage of maltodextrins up to at least twice that size is specifically mediated by maltoporin channels. The single channel conductance is four times lower than that of OmpF porin and is specifically inhibited by maltodextrins (Dargent et al., 1987). Moreover, maltoporin channels fluctuate with a unit step size, whereas in the case of OmpF, the closing step amounts to about one-third of the opening step. The maltodextrin-binding properties of maltoporin have been studied in some detail (Ferenci & Boos, 1980). Affinity measurements show that the binding of maltose itself is quite weak, with a dissociation constant in the millimolar range. Increasing chain length of linear maltodextrins drastically increases their binding affinity for maltoporin; thus, maltohexaose binds with a micromolar dissociation constant. The transport of maltose across the outer membrane is not an active process but proceeds strictly down a concentration gradient. There is, however, a scavenger protein in the periplasm that keeps the concentration of free maltose at micromolar levels and thus maintains net maltose influx. This protein is called the maltose-binding protein (MBP) and is encoded by the gene m&E. Its relative molecular mass is 40,099, and it exhibits tight binding of maltose and maltodextrins with similar affinities, all in the micromolar range (Szmelcman et al., 1976). It is known to interact with the MalFMalG-MalK complex that constitutes the active maltose transport system in the inner membrane (Shuman, 1982). On the basis of genetic evidence (Bavoil et al., 1983), it has always been assumed that MBP also binds to maltoporin, picking up a substrate molecule as it emerges from the pore, and subsequently dissociates from maltoporin to shuttle the substrate to the inner membrane (Wandersman et al., 1979). This model was given further support by a reported influence of MBP on maltoporin channel activity (Neuhaus et al., 1983). However, clear biochemical demonstration of such an interaction has
Protein Interaction
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proven difficult. Although immobilized MBP will bind maltoporin, this binding is not reversible even by high concentrations of maltose as would be expected (Bavoil & Nikaido, 1981). Analytical ultracentrifugation of maltoporin-MBP mixtures shows two bands corresponding to maltoporin trimer and MBP monomer, and fails to reveal the presence of a stable maltoporin-MBP complex (A. Lustig, K. A. Stauffer dz A. Engel, unpublished results). Furthermore, the stabilization of maltoporin conductance by MBP has not been reproduced (Dargent et al., 1987). Here, we report on MBP-maltoporin interactions studied by electron microscopy and image processing of 2-D crystals reconstituted from lipids, MBP and maltoporin. Substoichiometric binding is suggested by SDS/polyacrylamide gel electrophoresis of such arrays after extensive washing. A difference map from averaged unit cells of MBPmaltoporin arrays and an MBP-free control displays three symmetry-related MBP binding sites per maltoporin trimer. As assessed by multivariate statistical analysis, not more than one of these is likely to be occupied at a given time. The methods used here could be of general interest for the study of the structure of a soluble protein and its binding to an insoluble ligand reconstituted into a 2-D regular array. 2. Materials
and Methods
E. co5 W3110 were grown in M9 medium supplemented with 615% (w/v) Casamino acids and 92% (w/v) maltose.
Maltose-binding protein was purified from those cells according to the method of Ferenci Lb Klotz (1978). In some experiments, the maltose was eliminated by dialysis against 2 M-guanidium . HCl in 20 mna-Hepes (pH 7.4) against Hepes buffer alone. followed by dialysis Maltoporin was extracted from cells broken by passage through a French pressure cell and subsequently purified by a combination of anion-exchange chromatography, chromatofocussing and gel filtration as described (Stauffer et al., 1990). The final product migrated as a single band on SDS/polyacrylamide gel electrophoresis. No residual lipopolysaccharide could be detected by silver staining. OmpF porin was extracted from E. co& BE cells (strain BZB 3333) and isolated ss described (Holzenburg et al., 1989). Phospholipid (phosphatidyl ethanolamine, Sigma type V, from E. CA) was dried in a stream of nitrogen at 37°C and solubilized to either 1 or 10 mg/ml in buffer
from trimeric complexes and reveal unit cell dimensions of a = b = 7.9 nm. (a2) The correlation average, however, exhibits an apparent g-fold symmetry. This is due to 2 possible angular orientations around the 3-fold symmetry axis of the maltoporin trimers that differ by n. (a3) and (a4) The 2 correlation averages, which were calculated from 1 of the 2 layers using 2 different references, illustrate this point. (b) Rarely, tubes open up to grow out as single sheets that reveal a rectangular crystal habit as suggested by (bl) the diffraction pattern and (b2) the averaged projection. One unit cell contains 2 maltoporin trimers in opposite angular orientation and exhibits dimensions of a = 7.9 nm, b = 137 nm. Maltoporin monomers show a major and a minor domain separated by a groove. (c) The hexagonal pattern is distinctly visible on the micrograph of a double-layered maltoporin sheet (unit cell dimensions a = b = 94 nm). (cl) The optical diffraction pattern is quite different from that of vesicles, exhibiting strong (1,l) orders and no rotational misalignment. (~2) The averaged projection also reveals a different unit cell morphology from the other crystal forms, but it can be explained by a counter clockwise rotation of the maltoporin trimer by 12”. The scale bars on the micrographs represent 200 nm in (a) and (b), 100 nm in (c) and (20 nm)-’ in the diffraction patterns.
1158
K. A. Stauffer et al.
Figure 2. Reconstituted maltoporin-MBP arrays. (a) and (b) As without MBP, vesicles and tubes are found at LPR = O-1, while (c) multilayered sheets are formed at LPR = 1.0. (a) MBPs are randomly adsorbed to the free carbon film, whereas they appear to be partially ordered along the lattice lines of the single-layer maltoporin membrane. (al) The diffraction pattern indicates some distinct (2,2) orders, which correspond to a spatial frequency of approximately (2 nm)-‘. (a2) The average projection resembles that of the hexagonal maltoporin lattice without MBP (see Fig. l(a2)) and possesses an apparent 6-fold symmetry as well. (b) No ordered MBP decoration is discernible on maltoporin tubes, although randomly dispersed MBP is visible in the background. (bl) The diffraction pattern shows the top and bottom layer of the collapsed tube built from a rectangular maltoporin lattice. (b2) The average from 1 layer reveals 2 trimers per rectangular unit cell, similar to the average from the rectangular layer shown in Fig. l(b2). (c) Randomly arranged particles can be seen on the multilayered sheet reconstituted at LPR = 1.0. (cl) The diffraction pattern and (~2) the average closely match those from the MBP-free control. The scale bars on the micrographs represent 200 nm in (a) and (b), 100 nm in (c) and (2.0 nm)-’ on the diffraction patterns.
Maltopwin
Maltose-binding
Figure 3. Samples of coreconstituted MBP and maltoporin analysed on SDS/polyacrylamide gel electrophoresis. Lane a, membranes after reconstitution; lanes b to e, supernatants of 4 consecutive washes with MBP-free buffer; lane f, maltoporin-MBP membranes at the end of this washing procedure.
containing 7.5% octyl polyoxyethylene (octyl-POE, a kind gift from J. P. Rosenbusch). In a typical reconstitution experiment, 40 pl of maltoporin (10 mg/ml in buffer containing 1 y. octyl-POE) wss mixed with 40 ~1 of phospholipid solution, 280 ~1 of Hepes buffer (20 mM-Hepes (pH 7-O), 10 mna-M&Y,, lOO.mNaCl, 62 mr+r-EDTA, 3 mnr-NaN,, 02 mM-dithiothreitol) and 40 ~1 of MBP (10 mgJm1). The final maltoporin concentration was 1 mg/ml, and the molar ratio of MBP to maltoporin was slightly greater than 1. The ratio of phospholipid to maltoporin was either 61 or 1 (w/w). After passing the sample through a 645 pm Millipore filter it w&8 dialysed against a total of approximately 21 of Hepes buffer for 2 days in a continuous-flow apparatus as described (Engel et al., 1988). During the first 36 h the temperature was held constant at 37°C then it was decreased to 22°C in a linear ramp over 12 h. The association of MBP with different porin arrays was sssayed by SDS/polyacrylamide gel electrophoresis. A volume of 406 ~1 containing either O-4 mg of OmpF porin or O-4 mg of maltoporin coreconstituted with 04 mg of MBP in the presence of phospholipids was centrifuged in a Heraeus Biofuge A at 5000 revs/min for 5 min, the supernatant saved, and the pellet resuspended in 400~1 of Hepes buffer. This procedure ws.s repeated 4 times, but the pellet was resuspended in 40 ~1 of buffer after the last wash. A portion (10 ~1) of the initial solution, of each supernatant and of the resuspended pellet were applied to an SDS/l@5Oh polyacrylamide gel. Reconstituted protein-lipid membranes were adsorbed to electron microscope grids covered by carbon-topped formvar films that were rendered hydrophilic by glow discharge at low pressure in air. After washing in Tris buffer (10 mm-Tris. HCl, pH 7) the membranes were negatively stained with 075% (w/v) many1 formate (pH 42) ammonium molybdate (pH 66). or 2% (w/v) Alternatively, adsorbed membranes were washed in double-distilled water, frozen in liquid nitrogen, freezedried in a Balzer BAF 300 unit at -80°C and unidirectionally shadowed with tantalum/tungsten at an elevation angle of 45” at the same temperature. Images were recorded on Kodak SO-163 lilm at a nominal magnification of 50,000 x and a dose of approximately 2000 e/rim’ using a Hitachi H-7060 transmission
Protein
Interaction
1159
electron microscope operated at 100 kV. The magnification was calibrated using negatively stained oat&se crystals. Micrographs were evaluated for focus, astigmatism and drift as well as for crystallinity of reconstituted membranes by optical difliaction (see Aebi et oL, 1973). Suitable areas were digitized using an Eikonix 850 CCD camera equipped with a Zeiss S-planar objective lens. To eliminate noise introduced by gain and bias variations of the CCD-array, an empty frame was recorded and stored for on-line normalization. In addition, 3 x 3 pixel areas were averaged and stored as single pixels separated by @6 nm. Several methods were applied for calculating averaged projections of maltoporin unit cells with and without MBP, mainly taking advantage of the SEMPER image processing system (Saxton et aE., 1979). First, straightforward Fourier peak llltration (see Aebi et al., 1973) was used for a rapid evaluation of unit cell morphologies and crystal quality. Second, correlation averaging (Sexton & Baumeister, 1982) allowed the elimination of residual lattice disorder by locating the unit cells precisely using a reference that included 3 to 4 maltoporin trimers. In addition, correlation functions were used to discriminate 2 angular orientations of the maltoporin trimers with respect to the 3-fold axis. To increase the sensitivity, references were filtered by the clipped power spectrum of the difference map calculated from the 2 possible angular orientations. Third, subframes containing 4 trimers were extracted at the position of cross-correlation peaks and submitted to a multivariate statistical classification exercising a program package kindly provided by J. P. Bretaudiere (see Frank at al., 1988). The classification algorithm used “dynamic clouds” clustering for partitioning the images into 49 clusters and rejecting the images that could not be assigned to any of the 49 clusters. The clusters were then further partitioned by hierarchical sacending classification, yielding a clessification tree. Clusters or classes of MBP-decorated maltoporin trimers determined in this way were averaged and the difference images of such averages and maltoporin controls calculated. The weight of the control was adapted to minimize the dynamic power of the difference map. Surface reliefs were calculated from averages of freeze-dried, metal-shadowed maltoporin-membranes aa described (Smith & Kistler, 1977).
3. Results In agreement with a previous report (Lepault et al., 1988), maltoporin could be reconstituted into vesicles and tubes with E. coli phospholipids at a lipid-to-protein ratio (w/w; LPR) of 91 (Fig. l(a) and (b)). The packing arrangement of the maltoporin trimers is hexagonal in vesicles and rectangular in tubes. Top and bottom layers of collapsed vesicles were usually not in register, as visible from the diffraction pattern (Fig. l(a1)). The Fourier peak filtration of one layer locally revealed a p3 lattice (a = b = 79 nm) with significant irregularities (not shown), but a straightforward correlation average over a large area exhibited a rather puzzling p6 symmetry (Fig. l(a2)). A closer analysis, however, showed that maltoporin trimers were inserted in two distinct angular orientations that differed by n (Fig. l(a3) and (a4)). Such in-plane phase transitions involving rotation of trimers
K. A. Stauffer et al.
maltoporin arrays with a,& Figure 4. Correlation averages and multivariate statistical classification of different multiva without MBP. (a) (left) A 3-fold symmetry is evident in the average of 900 angularly oriented subframes containing 4 unit cells extracted from a single layer of a collapsed vesicle. The average calculated from 2500 MBP-decorated subframes is shown in (a) on the right side. (a) (centre) The difference map of these averages indicates the presence of additional mass in 3-fold related positions (1 to 3), the most likely binding sites of MBP. (b) Structural variations are characteristic for negatively stained aIT8yS. They are pronounced in collapsed vesicles as illustrated by averages of images not contained in clusters, images of the two major clusters, and images of the 3 major classes (from left to right). (c) Prominent particles become discernible on cluster-averages of MBP-decorated vesicles. They are localized at position 2 (1st row) or position 3 (2nd row) in the cluster-averages shown. (d) Difference maps calculated by subtrttction of the weighted control from individual cluster-averages were averaged after translational alignment (position 2: left; position 3: centre). Contours of the MBP projection delineating the edge and the core of the molecule, respectively, are drawn over the MBP-free maltoporin unit cell (right). The scale bars represent 5 nm.
Maltoporin
Maltose-binding
around their S-fold axis have been reported for OmpF porin (Dorset et al., 1989). Occasionally, tubes initiated the crystallization of sheets that revealed the large, single-layered rectangular unit cell (a = 7.9 nm, b = 13.7 nm) containing two maltoporin trimers (Fig. l(b)) with particular clarity. The projection of the rectangular unit cell displayed major and minor domains of the maltoporin monomer that were separated by a distinct groove. In general, the crystal order of negatively stained tubes and single layers (Fig. l(b)) was better than that of vesicles (Fig. l(a)), which were severely distorted as a result of flattening during negative staining/air drying. At LPR = 1, a new crystal habit was found. These large, double-layered hexagonal sheets (Fig. l(c)) with unit cell size a = b = 8.4 nm were well ordered as indicated by sharp diffraction spots extending to (2nm)-’ (Fig. l(c1)). The lack of rotational misalignment in these diffraction patterns suggested that the layers stacked in register, indicating a strong interaction between maltoporin trimers of different layers. Averaged projections showed trimeric pores that resembled those of the rectangular and hexagonal lattices (Fig. l(a3) and (b2)), but that were rotated by 24” about the central 3-fold axis of symmetry (Fig. l(c2)). This rotation was paralleled by a 13% increase in the unit cell area, thus providing space to accommodate additional lipids. All three crystal forms could be reconstituted in the presence of an excess of MBP, and for all three forms well-ordered lattices were observed (Fig. 2). A non-random position and orientation of membraneattached MBP was suggested by rows of particles along the direction of lattice lines on disrupted maltoporin vesicles (Fig. 2(a)). This is most distinct when the micrograph is viewed both at a glancing angle and along a lattice line. However, only a close comparison of the average in Figure 2(a2) with that in Figure l(a2) indicated a subtle reduction of the gap between subunits of adjacent pores, which could be the result of some additional mass. On tubes (Fig. 2(b)) and sheets (Fig. 2(c)), MBP particles were not deposited in an ordered fashion. Repeated washing of membranes reconstituted in the presence of MBP with equal buffer volumes revealed that MBP was membrane-bound, but that less than one MBP molecule per maltoporin trimer remained attached as suggested by the relative intensities of bands on SDS/polyacrylamide gel electrophoresis (Fig. 3). Controls with arrays from OmpF porin and dimyristoyl phosphatidyl choline incubated with MBP showed no binding. This very simple binding assay confirms that binding of MBP to maltoporin, under the conditions used in our reconstitution experiments at about 25 j.lM-protein, occurs in less than stoichiometric amounts. As illustrated by Figure l(a), maltoporin trimers were inserted in two distinct angular orientations into hexagonal vesicles. Therefore, individual pores, localized by their cross-correlation signal with a suitable reference (i.e. average in Fig. l(a2)), were
Protein Interaction
1161
extracted and their orientation determined by comparison with two references of opposite orientation (Fig. l(a3, a4)). Approximately half of the unit cells had to be rotated by 7~for subsequent averaging. The average of 900 maltoporin trimers generated in this way (Fig. 4(a), left) exhibited a stainpenetrated triangular pore similar to that of rectangular-tubes (Figs l(b) and 2(b)). The major and minor domains of maltoporin monomers were distinct and allowed the hand of the pore to be determined. The two maltoporin domains-were also visible on the average of 2590 maltoporin trimers from MBP-decorated disrupted vesicles (Fig. 4(a), right), but they appeared to be slightly larger than those of MBP-free vesicles. The difference map presented in Figure 4(a) (centre) indicated three prominent binding sites of MBP per trimer, which were located between adjacent pores. To separate MBP-free unit cells from those carrying one or several MBP molecules, oriented projections comprising four maltoporin trimers from decorated vesicles (Fig. 2(a)), tubes (Fig. 2(b)) and sheets (Fig. 2(c)) were submitted to multivariate statistical analysis (van Heel & Frank, 1981; van Heel, 1984; Frank et al., 1988). Bretaudiere’s classification algorithm (Frank et al., 1988) was used to determine clusters of similar images in a first step and to subsequently assess the similarity of different clusters by a hierarchical ascending classification scheme. This classification tree allowed clusters to be partitioned into classes of ascending order for subsequent averaging. Because this approach has been critically debated by van Heel (1989), we have tested its sensitivity and reproducibility with our own data. First, MBP-free maltoporin trimers were analysed before angular orientation. The algorithm divided unit cells into two major classes, the “up” and “down” orientations. In addition, we found that the ratio of up and down trimers was in excellent agreement with the result from correlation measurements. Second, refinement runs were done on various data sets, omitting images that could not be assigned to prominent clusters, thereby yielding a more pronounced clustering of the remaining images. The fact that we found only minor changes between averages calculated from images of a particular cluster during several refinement steps indicated appropriate functioning of the classification scheme (data not shown). As a next step, we analysed the variability of negatively stained, angularly aligned, MBP-free maltoporin trimers from single-layered arrays. As illustrated by Figure 4(b) (left), the average of projections comprising four pores that did not belong to any identified cluster (28.8% of the 900 cells analysed) exhibited a strong similarity to the undecorated pore calculated by correlation averaging (Figs l(a), right and 4(a), left). Variations due to stain irregularities and structural damage during dehydration were demonstrated by the averages of the two major clusters containing 47 y0 and 4.3 y0 of the cells, respectively, and the averages of the three
1162
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Figure 5. Freeze-dried, metal-shadowed maltoporin (a) tubes and (b) sheets show regular fine structures to a resolution of 2.5 nm (see (al)). Although reconstituted in the presence of MBP, no random or regular decoration of such surfaces occurs, whereas MBP molecules appear to adhere to the carbon film. (a2) and (a3) The surface relief reconstructed from the 2 tubes exhibit 2 trimeric depressions per rectangular unit cell, whereas (bl) and (b2) trimeric depressions are arranged in a hexagonal lattice at the surface of maltoporin sheets. The scale bars represent 50 nm in (a), 100 nm in (b) and (3.0 nm)-’ in (al).
Maltoporin
Maltose-binding
major classes (192%, 291% and 31.9%; Fig. 4(b), from left to right). In contrast to this, MBP-decorated maltoporin trimers in disrupted vesicles revealed outstanding features that suggested systematic binding of MBP. The first row in Figure 4(c) shows cluster-averages from a classification of 2500 areas containing four maltoporin trimers with one MBP bound at position 2 (see Fig. 4(a), centre), and the second row shows MBP in position 3. Two MBP molecules were bound in the vicinity of one pore quite frequently, but they consistently occupied sites of different maltoporin trimers (155%, data not shown), while less than 1 O/Oshowed more than two additional protuberances. In all, 296% of the 2500 areas analysed were not assigned to any clusters. Their average was indistinguishable from that of the MBP-free control, suggesting that about 30% of the unit cells were MBP-free. Since the shape of the bound MBP was obscured by the underlying maltoporin array, difference maps of all cluster-averages and appropriately weighted projections of MBP-free maltoporin trimers were calculated. Correlation functions between these difference maps and those showing the MBP in positions 1, 2 and 3, respectively (Fig. 4(a), centre) were used to refine the alignment of bound MBP for subsequent averaging. Maps exhibiting a correlation coefficient above 66 were then merged to calculate the MBP average for position 1, position 2 (Fig. 4(d), left), and position 3 (Fig. 4(d), centre). These averages were angularly aligned to produce the final MBP map containing 42% of the unit cells carrying at least one MBP. The contour of the MBP molecule was superimposed on the maltoporin trimer projection to illustrate the position of the MBP on the rim of the pore (Fig. 4(d), right). To further elucidate the interaction between MBP and maltoporin integrated in tubes and sheets, such membranes were freeze-dried and metal-shadowed (Fig. 5). The surfaces of both maltoporin tubes and sheets appeared to be very clean, with no traces of randomly or specifically bound MBP. Structural preservation to 25 nm (Fig. 5(al)) allowed the surface reliefs of both crystal habits to be reconstructed and the entrances of the pores to be visualized. As displayed in Figure 5(a2) and (a3), the tubes exhibited two triplet openings per rectangular unit cell, one of them rotated by n with respect to the other. Triplet openings arranged on a hexagonal lattice were clearly discernible on the surface of the sheets as well (Fig. 5(bl) and (b2)). Vesicles, on the other hand, were not well ordered enough for such an analysis. 4. Discussion The results presented here document binding of E. coli MalE protein, the maltose-binding scavenger located in the periplasmic space, to LamB protein, the maltoporin in the outer membrane. So far, no biochemical data are available that would allow this interaction to be characterized quantitatively,
Protein Interaction
1163
although conceptually, MBP-maltoporin binding is appealing, considering the functional aspects of the maltose transport system. Crosslinking experiments performed to show the existence of a MBPmaltoporin complex in solution (data not shown) have failed in our hands, and suggest that the presence of detergent, or the absence of lipid, may interfere with complex formation. We cannot assess at this point which of the two proteins is affected; it may be noted, however, that the binding of solubilized maltoporin to phage 1 seems to be somewhat impaired as well (Randall-Hazelbauer & Schwartz, 1973). With maltoporin reconstituted into a lipid bilayer, the MBP-maltoporin interaction becomes rather less intractable. The availability of wellordered reconstituted maltoporin membranes has tempted us to study the complex structurally, combining electron microscopy with digital imageprocessing. Indeed, we have been able to observe of maltoporin arrays with MBP, decoration although a majority of the possible MBP-binding sites appear to be unoccupied while some excess MBP remains at random positions. Fully induced bacteria are estimated to synthesize about 2 x lo4 molecules of MBP and up to 1 x lo5 molecules of maltoporin per cell (Schwartz, 1987). This corresponds to concentrations of around 50 PM over the whole cell. Moreover, since the distribution of both proteins is strictly limited to the periplasmic space and the outer membrane, respectively, local concentrations could be expected to reach several hundred PM. It is not surprising, therefore, that the interaction between the two proteins seems to require only a moderate affinity constant. On the other hand, when MBP binds a maltodextrin molecule, even a small conformational change in the maltoporin-binding domain of MBP can be expected to lead to dissociation of the complex if the binding affinity is not very high. MBP molecules properly bound to maltoporin arrays could be identified by correlation averaging and multivariate statistical analysis. Multivariate statistical classification (van Heel, 1989) separates images into subclasses according to a straightforward similarity criterion: it is indicated by the clustering of vectors in an N-dimensional space (where N = n x m) representing images containing n x m pixels. After extensive testing we have used Bretaudiere’s classification algorithm (Frank et al., 1988) to partition images of maltoporin trimers with and without MBP. This algorithm first determines the clustering of images by the dynamic cloud technique and then partitions the clusters by a hierarchical ascending classification scheme. The respective cluster-averages and class-averages of MBP-decorated maltoporin trimers were then further analysed for their angular orientation with respect to one another, and compared to averages from MBP-free controls. For single-layered hexagonal maltoporin lattices, this approach reproducibly yielded a distinct indication of three possible MBP-binding sites per malto-
1164
K.
A. Stauffer
porin trimer, of which not more than one appears to be occupied in most cases. No binding site could be identified on maltoporin tubes or double-layered sheets. Further investigation of tubes and double-layered sheets by freeze-drying and metal-shadowing produced an explanation of this finding: the surface reconstructions in Figure 5 show clearly that, at least in our hands, both tubes and double-layered sheets expose trimeric openings at their surfaces. In analogy to an assignment made for OmpF porin (Engel et al., 1985; Hoenger et al., 1990), we conclude this surface to be the extracellular face of maltoporin (as opposed to the assignment made by Lepault et al., 1988). MBP would therefore have to bind on the inner surface of narrow tubes, or between the layers of tightly interacting double sheets, a very unfavourable process in each case. The complete lack of MBP decoration on the outer surfaces of tubes would seem to support our conclusion about their sidedness. After aligning and averaging the most prominent cluster-averages containing 42% of the unit cells decorated with at least one molecule of MBP, the rough outline of the bound MBP becomes visible. The projection shows a triangular-shaped molecule with dimensions of approximately 5 x 4 nm. The X-ray structure (Spurlino et al., 1991) establishes MBP to be a triangular-shaped disk with dimensions of about 6 nm x 4 nm x 3 nm. Considering the similarity in the overall shape of the molecule, we propose that our map shows MBP in a projection with its shortest axis perpendicular to the plane of the membrane. Located for the greater part on the rim of the pore, the MBP molecule appears to project out over the pore and thus to occupy just the kind of position one would expect to be optimal for its interaction with incoming maltodextrins. It will be interesting to map the protein-protein interface on the X-ray structures of both MBP and, once it becomes available, maltoporin. The structural basis of interactions between integral membrane proteins and soluble proteins has not been studied in great detail so far, and the MBP-maltoporin pair might serve as a model system for other processes that involve the association and dissociation of a soluble protein from a membrane surface. We thank Jiirg Rosenbusch and Malcolm Page for stimulating discussions, Ueli Aebi and John Berriman for critical comments on the manuscript, and Andreas Hefti, Ariel Lustig and Ariane Hardmeyer for technical assistance. We are grateful to Jean-Pierre Bretaudiere for the programs for multivariate statistical analysis and classification. The work was supported by Swiss Kational Science Foundation grants 31-25684.88 to A.E., and 3.29485 to J. P. Rosenbusch, and the Maurice E. MiillerFoundation of Switzerland.
References Aebi,
U., Smith, P. R., Dubochet, J., Henry, C. & Kellenberger, E. (1973). A study of the structure of the T-layer of Bacillus brevis. J. Supramol. Struct. 1,
498-522.
et al. Bavoil, P. & Nikaido, H. (1981). Physical interaction between the phage I receptor protein and the carrierimmobilized maltose-binding protein of E. co& J. Biol. Chem. 256, 11385-11388. Bavoil, P., Wandersman, C., Schwartz, M. & Nikaido, H. (1983). A mutant form of maltose-binding protein of E. coli deficient in its interaction with bacteriophage 1 receptor protein. J. Bucteriol. 155, 919-921. Chalcroft, J. P., Engelhart, H. t Baumeister, W. (1987). Structure of the porin from a bacterial stalk. FEBS Letters,
211, 53-58.
Clement, J. M. & Hofnung, M. (1981). Gene sequence of the 1 receptor, an outer membrane protein of E. coli K12. Cell, 27, 507-514. Dargent, B., Rosenbusch, J. bz Pattus, F. (1987). Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E. coli outer membrane. FEBS Letters, 220, 136-142. Dorset, D. L., Massalski, A. K. & Rosenbusch, ?J. P. (1989). In-plane phase transition of an integral membrane protein: nucleation of the OmpF matrix porin rectangular polymorph. Proc. Nat. Acad. Sci., U.S.A. 86, 6143-6147. Engel, A., Massalski, A., Schindler, H., Dorset, D. L. & Rosenbusch, J. P. (1985). Porin channel triplets merge into single outlets in E. coli outer membranes. Nature
(London),
317, 643-645.
Engel, A., Holzenburg, A., Stauffer, K., Rosenbusch, J. & Aebi, U. (1988). A novel reconstitution method for inducing the formation of regular 2-dimensional arrays of membrane proteins and lipids. Proc. 46th Ann. Meeting Electron Microsc. Sot. Amer. (Bailey. G. W., ed.), pp. 152-153, San Francisco Press, San Francisco, CA. Ferenci, T. & Boos, W. (1980). The role of E. coli 1 receptor in the transport of maltose and maltodextrins. J. Supramol. Struct. 13, 101-116. Ferenci, T. & Klotz, U. (1978). Affinity chromatographic isolation of the periplasmic maltose binding protein of E. coli. FEBS Letters, 94, 213-217. Frank, J., Bretaudiere, J. P., Carazo, J. M., Verschoor, A. & Wagenknecht, T. (1988). Classification of images of biomolecular assemblies: a study of ribosomal subunits of E. coli. J. Microsc. 150, 99-115. Hengge, R. C Boos, W. (1983). Maltose and lactose transport in E. coli. Examples of two different types of concentrative transport systems. Biochim. Biophys. Acta, 737, 443-478.
Hoenger, A., Gross, H., Aebi, U. & Engel, A. (1996). Localization of the lipopolysaccharides in metalmembranes. shadowed reconstituted lipid-porin J. Struct. Biol. 103, 185-195. Holzenburg, A., Engel, A., Kessler, R., Manz, B. J., Lustig, A. & Aebi, U. (1989). Rapid isolation of OmpF porin-LPS complexes suitable for structurefunction studies. Biochemistry, 28, 4187-4193. Lepault, J., Dargent, B., Tichelaar, W., Rosenbusch, J. P., Leonard, K. & Pattus, F. (1988). Threedimensional reconstruction of maltoporin from electron microscopy and image processing. EMBO J. 7, 261-268. Keuhaus, J.-M., Schindler, H. & Rosenbusch, J. P. (1983). The periplasmic maltose-binding protein modifies the characteristics of maltoporin. channel-forming EMBO J. 2, 1987-1991. Randall-Hazelbauer, L. & Schwartz, M. (1973). Isolation of the bacteriophage lambda receptor from E. coli. J. Bacterial.
116, 1436-1446.
Saxton, W. 0. t Baumeister,
W. (1982). The correlation
Maltoporin
Maltose-binding
averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127-138. Saxton. W. O., Pitt, T. J. & Horner, W. (1979). Digital image processing: the SEMPER system.
Ultramicroscopy,
4, 343-354.
Schwartz, M. (1987). The maltose regulon. In Escherichia typhimurium (Neidhardt, F. C., coli and Salmonella ed.), vol. 2. pp. 148221502, American Society for Microbiology, Washington, DC. Shuman, H. A. (1982). Active transport of maltose in E. coli K12. J. Biol. Chem. 257, 54555461. Smith, P. R. & Kistler, J. (1977). Surface reliefs computed from micrographs of heavy metal-shadowed specimens J. Ultrastruct. Res. 61, 124-133. Spurlino. J. C., Lu, G.-Y. & Quiocho, F. A. (1991). The 23-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J. Biol. Chem,. 266. 520225219.
Edited
Protein
Interaction
1165
Stauffer, K. A., Page, M. G. P., Hardmeyer, A., Keller, T. A. & Pauptit, R. A. (1990). Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli. J. Mol. Biol. 211, 297-299. Szmelcman, S., Schwartz, M., Silhavy. T. J. & Boos, W. (1976). Maltose transport in E. coli K12. Eur. J. Biochem. 65, 13-19. van Heel, M. (1984). Multivariate statistical classification of noisy images (randomly oriented biological macromolecules). Ultramicroscopy, 13, 165-184. van Heel, M. (1989). Classification of very large electron microscopical image data sets. Opt&, 82, 114-126. van Heel, M. & Frank, J. (1981). Use of multivariate statistics in analyzing the images of biological macromolecules. Ultramicroscopy, 6, 187-194. Wandersman, C., Schwartz, M. & Ferenci, T. (1979). E. coli mutants impaired in maltodextrin transport.
.J.
by R. Huber
Bacterial.
140,
l-13.
