Research Article 127

Crystal structures of various maltooligosaccharides bound to maltoporin reveal a specific sugar translocation pathway R Dutzler1, Y-F Wang 2, PJ Rizkallah 3, JP Rosenbusch 2 and T Schirmer1*

Background: Maltoporin (which is encoded by the IamB gene) facilitates the Addresses: Departments of lStructural Biology translocation of maltodextrins across the outer membrane of Ecoli. In particular, and 2Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland it is indispensable for the transport of long maltooligosaccharides, as these do and 3Daresbury Laboratory, Daresbury, not pass through non-specific porins. An understanding of this intriguing Warrington, Cheshire WA4 4AD, UK. capability requires elucidation of the structural basis. *Corresponding author. Results: The crystal structures of maltoporin in complex with maltose, Key words: guided diffusion, LamB, maltodextrin, maltotriose and maltohexaose reveal an extended binding site within the membrane protein, X-ray structure maltoporin channel. The maltooligosaccharides are in apolar van der Waals contact with the 'greasy slide', a hydrophobic path that is composed of aromatic Received: 13 Oct 1995 Revisions requested: 6 Nov 1995 residues and located at the channel lining. At the constriction of the channel the Revisions received: 24 Nov 1995 sugars are tightly surrounded by protein side chains and form an extensive Accepted: 4 Dec 1995 hydrogen-bonding network with ionizable amino-acid residues. Structure 15 February 1996, 4:127-134 Conclusions: Hydrophobic interactions with the greasy slide guide the sugar © Current Biology Ltd ISSN 0969-2126 into and through the channel constriction. The glucosyl-binding subsites at the channel constriction confer stereospecificity to the channel along with the ability to scavenge substrate at low concentrations.

Introduction path that extends from the floor of the vestibule, through The presence of a porin in a Gram-negative bacteria the constriction, to the peri plasmic outlet. At low resolu­ implies that a large, water-filled channel spans the bacter­ tion (4 A) it has been demonstrated that maltotriose binds ial outer membrane, allowing small and polar molecules to this 'greasy slide' at the level of the constriction [4]. In to pass across the barrier. More specific porins, such as this report, we describe in detail the binding modes of maltoporin from E. coli, also facilitate the diffusion of maltodextrins of various lengths to maltoporin, as revealed large solutes such as maltodextrins, the starch degradation by X-ray structure analysis. The elucidation of glucosyl­ product (for reviews see [1,2]). At least seven a1-4linked binding subsite structures allows the translocation process glucosyl units can pass through the pore [3], despite its of maltooligosaccharides through maltoporin channels to small diameter (determined as 5 A in the recent crystal be discussed. sirllcture analysis [4]). This seems quite remarkable, as the deduced dimensions of highly selective ion channels Results are in the same range [5]. The maltoporin channel dis­ Complexes of maltoporin with maltose (Glcz), maltotriose criminates, mainly on the oligosaccharide level, between (Glc3), and maltohexaose (Glc6), were prepared by soaking sugars [6] and is indispensable for the translocation of native crystals in sugar solutions at concentrations well long maltooligosaccharides (larger than three glucosyl above their respective K[) values. The crystalline order moieties) as these do not pass through non-specific did not suffer upon this treatment and complete X-ray dif­ porins [71. Binding and ion inhibition studies [8-11] fraction data sets were collected (Table 1). The variation have demonstrated that, within the channel, there is a in the resolution of the data sets is mainly due to the dif­ maltooligosaccharide-binding site and that sugar binding ferent radiation sources used. The data quality was limited inhibits ion flow. by the fragility of these membrane protein crystals and by the weakness of the diffraction spots due to the large size The homotrimeric structure of maltoporin [4] exhibits of the unit cell. However, the threefold structural redun­ three independent channels that emanate from a common dancy within the asymmetric unit and the high solvent vestibule at the extracellular surface. The scaffold of the content of the crystals (76%) allowed substantial improve­ monomer is an IS-stranded antiparallel 13 barrel (Fig. 1a). ment of the maps and elimination, by cyclic averaging, of Three loops from the end of the barrel that is exposed at any model bias (Table 2). There was no indication of the cell surface are folded inwardly and contribute to a deviation from the local symmetry and refinement of the mid-channel constriction (Fig. Ib). Six contiguous aro­ models was performed with strict non-crystallographic matic residues line the channel and form a hydrophobic symmetry constraints. 128 Structure 1996, Vol 4 No 2

Figure 1

(a)

The fold of the maltoporin monomer [4]. (a) Stereo view of the Co: vertical axis. Loop L3 that is forming part of the channel constriction is trace with every 20th residue labelled. The threefold axis (not shown) coloured red. The aromatic residues of the greasy slide that constitute of the trimeric structure runs vertically in front of the monomer. The cell a continuous apolar path at the channel lining and Tyr118 (located at exterior is at the top and the periplasmic space is at the bottom. the opposite site of the channel) are shown in ball representation and (b) Ribbon representation [34] of the monomer fold . With respect to are coloured in green. the view in (a) the model has been rotated by about -90° around a

Table 1 Table 2

X-ray diffraction data. Cyclic averaging and refinement statistics.

Maltooligosaccharide in the Maltooligosaccharide in maltoporin complex Glc Glc Glc 2 3 s the maltoporin complex Glc2 Glc3 Glce X-ray source synchrotron lab source synchrotron Resolution* (A) 2.6 3.2 2.8 Number of crystals 3 2 3 Real space correlation 0.84 0.96 0.89 Resolution range (A) 30-2.6 30-3.2 30-2.8 Rave/ (Ofo) 14.1 12.6 15.9 Unique reflections 81651 51209 68475 (Ofo) 22.6 21.7 23.4 9.1 (34.5) 9.5 (35.1) 8.3 (37.0) ~aet o ,* Rmerge (Ofo) 24.3 23.4 25.3 Completeness (Ofo) 89.2 (90.9) 99.2 (99.8) 94.3 (95.3) R'ree§ (Ofo) Rmsd from ideal values Redundancy 3.6 (3 .1) 3.7 (3 .6) 3.0 (3.2) bond lengths (A) 0.018 0.017 0.020 Values given in parenthesis are for the highest resolution shell with an bond angles (0) 2.1 2.2 2.2 approximate width of 0.1 A. All data were collected on image plate detectors Number of water molecules 167 108 (MARRESEARCH) with a diameter of 300 mm. The crystals are of space­ Mean B value (A2) 34.7 28.0 43.0 group C2221 with cell constants that are identical, to within 1.5 A, with those of native maltoporin crystals (a=131 .9 A, b=214.8 A, c =220.2 A; [4]). *The low resolution cut-off was 15 A for the cyclic averaging procedure and 8 A for the refinement. tRaver is the averaging R factor (Fe calculated from the final averaged map) . *R'aetor is the conventional The structures of maltoporin in the complexed forms R factor (Fe calculated from final model). §~ree is the R factor superimpose well with native maltoporin. The root mean calculated with 100f0 (50f0 for the Glc3 complex) of the data that were not used for refinement. square (rms) deviations of the ea positions are all below 0.35 A and there are no significant differences in the back­ bone traces. Also, the amino-acid side chains of the channel discussion, the glucosyl-binding subsites that were lining that are in direct contact with the sugars in the com­ revealed by the structure analyses are numbered from 81 plexed forms show little variation in their structure (84 to 84 (starting the count from the channel vestibule). The atoms superimpose with a nTIS deviation of less than 0.6 A). bound glucosyl moieties are labelled g1 to g4 according to the subsites they occupy. For all three sugar soaks, Fa-Fe difference maps clearly revealed the presence of localized sugars within the The maltoporin-GI~ complex channel and the cyclic-averaged electron-density maps Elongated residual density (Fig. 2c) extending to lie in (Fig. 2) allowed detailed interpretation. In the following close proximity to that of the 'greasy slide', is found for Research Article Maltoporin-maltooligosaccharide complexes Dutzler et al. 129

Figure 2

Parts of the maltoporin complex models with superimposed cyclic averaged electron density maps. The sugar models have not been used for phasing of the maps that went into the averaging procedure. (a) Maltoporin-Glc6 complex. Cross-section through the channel at the height of the constriction, viewed from the extracellular side. The map has been contoured at 1.5

Table 3 T he 'upper' G lcz molecul e (glucosyls g l and gZ) is in apolar va n der Waals contact with the end of the greasy Hydrophobic interactions between the glucosyl moieties of maltooligosaccharides and the greasy slide of maltoporin. slide that exte nds in to the channel vestibule, that is, .with T rp#74 (a resid ue donated by an adj acent monome r of the gl g2 g3 g4 trime ri c structure) and Tyr41 (Fig. 3, Table 3). It is also in Trp#74 3.8 contact with Tyr11 8 of the opposite channel lining. A good fit with the e lectron density is ac hieved onl y for a maltose mode l that has its non-reducing end (gZ) pointing Tyr41 3.5 3.9 towa rd s the channel constriction (Fig. Zc). All hydroxyl 4.5 3.7 groups of this leading glu cosyl moiety are engaged in 4.1 3.3 hydrogen bonds. T he OZ-H hydroxy l inte racts with the Tyr6 3.8 3.6 03-H hyd roxyl group of the preceding sugar res idue. T he 3.7 3.2 re maining hydroxy l groups are bound to protein residues 3.7 3.5 Arg8, As pl16, and Arg8Z (Figs 4a, 5a). Glucosyl moiety g l is stacked upon T rp#74 and does not form hydrogen Trp420 3.8 3.6 bonds with the protei n. 3.5 T he ' lower' G lcz molecul e (glucosyls g3 and g4) is in Group B factor (A2) 37 39 38 34 apolar va n der Waals co ntact with th e central part of the 81 47 54 94 51 57 greasy slid e (Tyr41, Tyr6, T rp4Z0; F ig. 3, Table 3) and obstructs the channe l constricti on. F ive out of the eight The distances (A) of closest approach between apolar atoms are given hydroxyl groups of this G lcz molecul e are in volved in in matrix form. The three numbers of each entry refer, from top to hydrogen bonds with amino-acid sid e chain s (Figs 4a,5a). bottom, to the glucosyl moieties of the Glc2, Glc3 and Glc6 complex, respectively. The cut-off distance is 4 A. Group B factors of the Again, the re is an intramolecul ar hydrogen bond glucosyl moieties are given in the last row. between th e two glucosyl residues, whi ch is charac­ teri sti c of the he li cal conform ati on of maltodextrins. the crys tal soak with Glcz. At lower conto uring level (0.80') Resid ue Tyr11 8 points its hyd roxyl group towa rd s 0 4 of additional density located at va n der Waa ls di stance to the the glycosidic li nk between g3 and g4 (a ltho ugh the pe ri plas mic end of the greasy slide (Trp358) becomes distance of 3.5 A is too large fo r th e form ation of a strong visible. Only the major density has been modell ed. T he hydrogen bond). two consecutive Glcz molecules (Fig. Zc) overl ap sli ghtly and therefore have been assigned an occupancy of 0.5 The complexes of maltoporin with Glc3 and Glcs each. The group B factors are similar for all four glucosyl T he electro n density of the G lc} compl ex (not shown) moieti es (T able 3). as well as of the G lc6 complex (Fig. Zb) shows three

Figure 3

Stereographic superposition of the maltooligosaccharide molecules as determined in complex with maltoporin (after structural alignment of the residues of th e maltoporin channel lining) . The view is about

the same as in Fig. 1 b. Th e two Glc2 are shown in light blue, the Glc3 model is coloured red and the partial model of Glc6 is drawn in green. Also shown are the aromatic residues of the greasy slide (re sidue Trp#74 is donated by an adjacent subunit) and residue Tyrl18. Research Article Maltoporln-maltoollgosaccharlde complexes Dutzler et al. 131

Figure 4

Stereo views of maltooligosaccharide molecules bound to maltoporin. The view is approximately perpendicular (with the channel axis vertical) to the view shown in Figure 3. Hydrogen bonds (length < 3.2 A) are shown as thin cyan lines. (8) Maltoporin-Glc2 complex. The 'upper' Glc2 molecule is composed of glucosyls gl and g2, the 'lower' molecule of g3 and g4. The aromatic residues shown in cyan constitute part of the greasy slide and are, from the top, Trp#74, Tyr41 , Tyr6 and Trp420. (b) Maltoporin-Glc6 complex structure showing the three well­ defined glucosyl residues (g2 to g4, from the top). The aromatic residues shown in cyan are, from the top, Tyr41 , Tyr6 and Tyr420.

well-ordered glycosyl moieties at the constrictIOn of the Both sugars are in a left-handed helical conformation. channel. The chain directionality of the maltodextrins, Residues g3 and g4 of the Glc3 and of the Glc6 models with the non-reducing end pointing towards the periplas­ superimpose closely with the corresponding residues of mic outlet of the channel, is well defined. For the Glc6 the Glcz complex (Fig. 3). They are tightly bound to the complex, extension of the electron density towards the protein by the same set of interactions (Figs 4b, Sb) that channel outlet becomes visible when contoured at lower have been described in the previous paragraph. Residues level (Fig. Zb). gS and g6 model the weak density found in the Glc6 soak.

Figure 5

Schematic view of the polar interactions between oligosaccharides and maltoporin: (8) Glc2; (b) Glc6. Possible hydrogen bonds are drawn as broken lines and their lengths (A) refer to the distances between the involved heavy atoms. The glucosyl moieties are labelled according to the subsite that they occupy. 132 Structure 1996, Vol 4 No 2

These residues continue the helix defined by the preceding This would be due to the mismatch between the helix residues and are, therefore, detached from the greasy slide. curvatures of maltooligosaccharides [15] and the greasy slide. Tyr118 appears to serve as a steric barrier, only The variation between the complexes of the glucosyl allowing the passage of near-planar substrates such as orientations in position g2 (between Tyr41 and Tyr118; sugars composed of glucosyl residues. Fig. 3) is noteworthy. In the Glcz complex, part of this sugar residue is in apolar contact with Tyr41; in the Glc6 What is the reason for the variation in the orientation of complex, the glucosyl moiety is almost equidistant from glucosyl g2 as observed in the three complex structures both tyrosine neighbours; and in the Glc3 complex, it is in (Fig. 3)? Glucosyl g2 in maltotriose is the trailing end of apolar contact with Tyr118 (Table 3). The shape of the this sugar and is free to move towards Tyr118 and attach electron density for g2 of Glc6 indicates the presence of a to this residue by virtue of apolar interactions. Conversely, second (minor) conformation which would supenmpose glucosyl g2 of the Glcz complex is the leading residue of approximately with g2 of the Glc3 complex. the 'upper' maltose molecule. Its free 04-H hydroxyl is involved in a hydrogen bond to Asp116 and the residue is It is striking that six out of the seven polar protein ligands constrained by the link to gl which, in turn, is attached to to the sugars are charged (Fig. 5). At each of the two long Trp#74. Finally, the conformation of glucosyl g2 of Glc6 edges of the maltooligosaccharides the same pattern of a should represent that of a glucosyl residue at position 82 carboxylate group between two guanidinium groups is of a long maltooligosaccharide that still has its trailing end found (Arg8, Asp116, Arg33; Arg82, Glu43, Arg109). The in the vestibule. The observed variability in the g2 orien­ distances between these charged residues are too large for tation probably reflects the increased amount of accessible the formation of salt bridges. space at the entrance of the channel compared with the situation at the channel constriction where glucosyl Discussion moieties g3 and g4 are tightly confined. The architectures of non-specific porins involve a constric­ tion half-way across the membrane [12,13] and this struc­ The polar interactions between protein and sugars almost ture is encountered again in maltoporin. The hourglass exclusivcly involve ionizable residues, a binding mode shape of the non-specific pores allows maximal diffusion that causes strengthening of the hydrogen bonds and that rates across the membrane, while maintaining the size and has also been observed for other complexes [16]. Three of ion selectivity of the pore. the four arginine residues that bind to the sugars are struc­ turally stabilized by salt bridges with juxtaposed acidic In maltoporin, the constriction is, in addition, tailor-made residues (Arg8-Glu39, Arg33-Glu37, Arg82-Asp#73). This for the binding of the substrate, conveying specificity to probably contributes to the observed rigidity of the this channel. As a consequence, the substrate concentra­ channel lining and may explain the observation that no tion is enriched at the narrowest part of the channel and significant alterations in side-chain conformation are the translocation rate is enhanced at low substrate concen­ observed upon sugar binding. tration, that is, at concentrations below the Ko [14]. This allows the bacteria to uptake nutrients efficiently from the Point mutants of maltoporin that show altered starch affin­ external medium. ity have been selected [17,18] and analyzed thoroughly by the techniques of liposome swelling [19] and ion-flow The structure of the maltoporin channel appears to meet inhibition [20]. The present study reveals that all but one nicely the requirements for the binding of a maltooligosac­ of the affected residues (Arg8, Trp74, Arg82, Tyr118, chari de molecule in the left-handed helical conformation Asp121) interact directly with maltodextrins. Thus, the predominantly present in solution [15]. Its channel lining selection scheme employed was remarkably successful in matches the amphipathic nature of saccharides with the identifying residues responsible for substrate binding. rather apolar pyranose ring and the equatorial hydroxyl Only Asp121 shows no direct interaction with a sugar; groups. From the Glc6 complex structure it was possible to however this residue, appears to be important for main­ deduce that maltoporin provides well defined binding taining the channel constriction structure, as its side-chain subsites (82, 83, 84) for three consecutive glucosyl is hydrogen bonded to the main-chain of loop L3 and residues. It should be considered that the electron density makes a lateral contact with Tyr118. Of all mutants, only of the Glc6 soak probably represents a composite of all the Tyr118-+Phe mutation shows an increase in binding possible binding modes that involve the occupation of affinity for maltodextrins, in particular Glc3 [20]. This subsites 82 to 84. The remaining glucosyl units thus pro­ emphasizes the importance of the apolar contact between trude into the vestibule and the channel outlet. It seems Tyr118 and the sugar that is observed in the Glc3 complex. likely that longer maltooligosaccharides also do not attach to the entire length of the greasy slide, but only to the From binding and ion-inhibition studies with various three central aromatic residues at the channel constriction. maltooligosaccharides it has been concluded that the Research Article Maltoporin-maltooligosaccharide complexes Dutzler et al. 133

maltoporin channel contains three to five glucosyl-binding The series of complex structures determined in this study subsites [9,21]. This seems to be consistent with the has outlined three sites that are likely to be occupied observed complex structures described here that show three sequentially by the glucosyl residues of a maltooligosac­ well defined glucosyl-binding subsites. However, for charide molecule during translocation. The structures will cntropic reasons, an increase of the free binding energy with serve as a comprehensive basis for future simulations of sligar length may be expected for sugars larger than three sugar translocation by molecular dynamics. residues, because more binding states become accessible. Biological implications Binding of maltodextrins with lengths of up to ten gluco­ Maltoporin, an outer membrane protein from E. coli, syl units has been demonstrated [22], but, presumably, carries out the formidable task of allowing the there is no upper length limit. Indeed, the channel is large translocation of linear oligosaccharide molecules enough at its vestibule and outlet to accommodate a long across the membrane barrier. The process is driven maltooligosaccharide helix (defined by 02-H ... 03-H by the solute concentration gradient and shows speci­ hydrogen bonds between adjacent sugar residues) that ficity with respect to maltooligosaccharides, the degra­ is bound at the channel constriction in the manner dation products of starch. The maltoporin gene observed for Glc6. (lamB) is part of the maltose regulon which codes for all components necessary for transport and degrada­ One of the first studies of sugar translocation by malto­ tion of maltooligosaccharides, including the peri­ porin revealed a marked specificity that became most pro­ plasmic maltodextrin-binding protein [24]. nounced for long oligosaccharides [6]. To a large extend this may reflect the specificity of the sugar-binding site. The three-dimensional structures of maltoporin in From the analysis of sugar-induced current noise observed complex with maltooligosaccharides of various for maltoporin inserted into artificial bilayers, Benz and lengths have revealed the details of a specific sugar­ coworkers [23] have derived kinetic parameters for mal­ binding site. The efficiency of the channel is based on tooligosaccharide binding. Interestingly, the rates of asso­ the location of the binding site at the channel con­ ciation are found to be approximately independent of the striction and on the presence of a hydrophobic path at sugar length, whereas a marked decrease in the dissocia­ the channel lining that guides the substrate to the tion rate accompanies increase in sugar length. This obser­ peri plasm through the constriction. The channel can vation fits the notion that threading of the sugar into the be regarded as an enzyme that catalyses the trans­ channel is not dependent on its trailing end. location of a substrate from one compartment to another [25]. In response to the external (low) sugar We have proposed a guiding function for the greasy slide concentration this system probably has been opti­ for maltooligosaccharides [4]. At the start of translocation, mized with respect to its kca/KM ratio. It is likely that the hydrophobic patches of glucosyl moieties would inter­ the general structural principles found for maltoporin act with the end of the greasy slide that is extending into will be encountered again for other passive transport the channel vestibule (Trp#74 and Tyr41) and which is systems such as ion channels [26]. easily accessible. The affinity of this part of the hydropho­ bic path for glucosyl residues is revealed by the Glcz Material and methods complex structure. For further translocation the leading Purification, crystallization and crystal soaking sugar residue has to tilt towards Tyr41, that is, into an ori­ Native maltoporin was purified and crystallized as described [27]. entation as observed in the Glc6 or Glc3 complex. This Complexes of maltoporin with three different maltodextrins were obtained by crystal soaking. To this end crystals were transferred would enable the sugar to slide through the pore, via posi­ to a sugar solution which contained 50 mM maltose, 10 mM tions g3 and g4, towards the channel outlet. maltotriose, and 5mM maltohexaose, respectively, solubilized in the standard storage buffer (20mM N·2-hydroxymethylpiperazine-N'-2- After insertion into the channel, long maltooligosaccha­ ethanesulfonic acid [Hepes], pH7, 0.4% i3-decylmaltoside, 0.1 % rides would bind to subsites g2 to g4 in the manner dodecyl-nonaoxyethylene [C12E9], 0.1 M MgCI 2, 3mM NaN3 and 15% polyethylene glycol 2000). The soaking time was about 48 h observed for the Glc complex and perform a one-dimen­ 6 in each case. sional diffusion. Due to the helical arrangement of the subsites, translocation would proceed in a screw-like X-ray data collection and processing fashion, with retention of the helical structure of the sugar, Diffraction data for the maltodextrin complexes were collected at the very much like the movement of a bolt through a nut. synchrotron beamline BW6 at DESY, Hamburg (Glc6 complex), at a During this process no large energy barriers would have to ELLIOTT GX20 rotating anode generator (Glc3 complex), and at the be overcome because of the smooth surface of the greasy 9.6 wiggler synchrotron beam line at Daresbury (Glc2 complex). Data slide and the multitude of polar groups at the channel con­ were processed using the programs REFIX and MOSFLM [28,29]. Programs ROTAVATA/AGROVATA of the CCP4 package [30], were striction that would allow almost continuous exchange of used for scaling and merging of the data. Data collection and reduction hydrogen bonds. statistics are summarized in Table 1 . 134 Structure 1996, Vol 4 No 2

Structure solution and refinement 11. Gehring, K., Cheng, C.-H., Nikaido, H. & Jap, BK (1991). The asymmetric unit of the crystals contains three monomers that are Stoichiometry of maltodextrin-binding sites in LamB, an outer related by a local triad. Complex structure determination was initiated by membrane protein from Ecoli. J. Bacterial. 173, 1873-1878. 12. Cowan, S.W., et a/., & Rosenbusch, J.P. (1992). Crystal structures rigid-body refinement (three independent monomers) of the native mal­ explain functional properties of two E coli porins. Nature 358, 727-733. toporin model [4] against the respective diffraction data using the 13. Weiss, M.S. & Schulz, G.E. (1992). Structure of porin refined at 1.8 A program XPLOR [31]. The resulting model was used for derivation of resolution. J. Mol. Bioi. 227, 493-509. the local symmetry operators and for calculation of a 8igmaA weighted 14. Death, A, Notley, L. & Ferenci, T. (1993). Derepression of LamB protein (2Fo-Fe, Cl. e) map with programs of the CCP4 package [30]. Phase facilitates outer membrane permeation of carbohydrates into Escherichia refinement was performed by cyclic averaging with intermittent updating coli under conditions of nutrient stress. J. Bacterial. 175, 1475-1483. of the local symmetry operators using the program package RAVE [32]. 15. Goldsmith, E., Sprang, S. & Flettrick, R (1982). Structure of maltoheptaose by Difference Fourier methods and a model of The sugar models were fitted into the final averaged map using the glycogen. J. Mol. Bioi. 156, 411-427. 16. Spurlino, J.C., Lu, G.-Y. & Quiocho, FA (1991). The 2.3 Aresolution graphic program 0 [33]. The complex models were refined by conven­ structure of the maltose- or maltodextrin-binding protein, a primary tional positional refinement [31] with strict non-crystallographic symme­ receptor of bacterial active transport and chemotaxis. J. Bioi. Chern. try constraints. Restrained refinement of atomic B factors (with target 266,5202-5219. standard deviations for bonded main-chain and side-chain atoms of 17. Ferenci, T. & Lee, K.-S. (1982). Directed evolution of the lambda 2 A2 and 3 A2, respectively, and of 3 A2 and 4 A2 for atoms connected receptor of Escherichia coli through affinity chromatographic by a bond angle) was performed for complexes Glc2 and Glc6. For the selection. J. Mol. Bioi. 160, 431-444. 18. Heine, H.G., Kyngdon, J. & Ferenci, T. (1987). Sequence determinants Glc3 complex, individual B factors were taken from the Glc6 complex and only the overall B factor was refined. The atom occupancies of the in the IamB gene of E coli influencing the binding and pore selectivity of maltoporin. Gene 53, 287-292. two Glc molecules were set to 0.5 each, as they represent alternative 2 19. Nakae, T., Ishii, J. & Ferenci, T. (1986). The role of the maltodextrin­ binding modes. The atom occupancies of glucosyls g5 and g6 of the binding site in determining the transport properties of the LamB Glc6 soak were set to 0.75 and 0.5, respectively, according to their protein. J. Bioi. Chern. 261, 622-626. statistic weight under the assumption that all possible binding modes 20. Benz, R, Francis, G., Nakae, T. & Ferenci, T. (1992). Investigation of of the hexaose to the three consecutive subsites 82 to 84 are occu­ the selectivity of maltoporin channels using mutant LamB proteins: pied equally. As the resolution of the Glc3 complex structure did not mutations changing the maltodextrin binding site. Biochem. Biophys. permit refinement of atomic B factors, a grouped B factor was refined Acta 1104, 299-307. for the glucosyl moieties of all sugars in the final round in order to allow 21. Ferenci, T. (1989). Selectivity in solute transport: Binding sites and channel structure in maltoporin and other bacterial sugar transport comparative analysis. For the Glc2 and Glc6 complex models, water proteins. BioEssays 10, 3-7. molecules were incorporated if they showed Fo-Fe density above 4cr 22. Ferenci, T., Schwentorat, M., Ullrich, S. & Vilmart, J. (1980). Lambda and had reasonable hydrogen-bonding partners. Water molecules with receptor in the outer membrane of Escherichia coli as a binding refined B factors above 80 A2 were removed subsequently. protein for maltodextrins and starch polysaccharides. J. Bacterial. 142, 521-526. The coordinates of the maltoporin-maltooligosaccharide complexes 23. Andersen, C., Jordy, M. & Benz, R (1995). Evaluation of the rate are being deposited in the Brookhaven Protein Data Bank, for release constants of sugar transport through maltoporin (LamB) of E coli from one year from the date of pUblication. the sugar-induced current noise. J. Gen. Physiol. 105, 385-401 . 24. Schwartz, M. (1987). The maltose regulon. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. (Neidhardt, Acknowledgements F.C., ed), pp. 1482-1502, American Society of Microbiology, We thank HD Bartunik from the AGSM of the Max-Planck-Gesellschaft for Washington, D.C. providing the data collection facility at the synchrotron DESY (Hamburg). 25. Eisenberg, RS. (1990). Channels as Enzymes. J. Membr Biol.115, Support by the Swiss National Science Foundation (grants to TS and JPR) 1-12. is gratefully acknowledged. 26. MacKinnon, R (1995). Pore loops: an emerging theme in ion channel structure. Neuron 14, 889-892. References 27. Keller, TA, Ferenci, T., Prilipov, A & Rosenbusch, J.P. (1994). 1. Nikaido, H. & Vaara, M. (1985). Molecular basis of bacterial outer Crystallization of monodisperse maltoporin from wild-type and mutant membrane permeability. Microbiol. rev. 49, 1-32. strains of various Enterobacteriaceae. Bioch. Biophys. Res. Commun. 2. Nikaido, H. (1992). Porins and specific channels of bacterial outer 199,767-771. membranes. Mol. Microbial. 6, 435-442. 28. Kabsch, W. (1993). Automatic processing of rotation diffraction data 3. Wandersman, C., Schwartz, M. & Ferenci, T. (1979). E coli mutants from crystals of initially unknown symmetry and cell constants. J. Appl. impaired in maltodextrin transport. J. Bacteriol. 140, 1-13. Crystal. 26, 795-800. 4. Schirmer, T., Keller, TA, Wang, Y.-F. & Rosenbusch, J.P. (1995). 29. Leslie, AG.W. (1991). Recent changes to the MOSFLM package for Structural basis for sugar translocation through maltoporin channels at processing film and image plate data. In Joint CCP4 and 3.1 Aresolution. Science 267, 512-514. ESF-EACBM Newsletter on Protein Crystallography. SERC 5. Dwyer, T.M., Adams, D.J. & Hille, B. (1980). The permeability of the Daresbury Laboratory, Warrington, UK. endplate channel to organic cations in frog muscle. J. Gen. Physiol. 30. Collaborative Computational Project Number 4 (1994). The CCP4 suite: 75, 469-492. programs for protein crystallography. Acta Cryst. D 50, 760-763. 6. Luckey, M. & Nikaido, H. (1980). Specificity of diffusion channels 31. BrUnger, AT. (1992). X-PLOR Manual, Version. 3.1, Yale University, produced by lambda phage receptor protein of Escherichia coli. New Haven, CT. Proc. Natl. A cad. Sci. USA 77, 167-171. 32. Kleywegt, G.J. & Jones, T.A (1994). Halloween ... masks and bones. 7. Benson, SA, Occi, J.L.L. & Sampson, BA (1988). Mutations that In Proceedings of the CCP4 study weekend: From first map to final alter the pore function of the OmpF porin of Escherichia coli K12. model, (Bailey, S., Hubbard, R & Waller, D. eds), pp. 59-66, SERC J. Mol. Bioi. 203, 961-970. Daresbury Laboratory, Daresbury, UK. 8. Benz, R, Schmid, A, Nakae, T. & Vos-Scherperkeuter, G.H. (1986). 33. Jones, T.A & Kjeldgaard, M. (1993). 0 Version 5.9, The Manual. Pore formation by LamB of Escherichia coli in lipid bilayer Uppsala University, Uppsala, Sweden. membranes. J. Bacterial. 165, 978-986. 34. Kraulis, P.J. (1991). MOLSCRIPT: a program to produce both detailed 9. Benz, R, Schmid, A, Greetje, H. & Vos-Scheperkeuter, H. (1987). and schematic plots of protein structures. J. Appl. Crystal. 24, 946-950. Mechanism of sugar transport through the sugar-specific LamB channel of Escherichia coli outer membrane. J. Membr. Bioi. 100, 21-29. 10. Dargent, B., Rosenbusch, J.P. & Pattus, F. (1987). Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of Ecoli outer membrane. FEBS Lett. 220, 136-142.