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Chapter 26 Ion selectivity and substrate specificity: The structural basis of porin function
CHAPTER 26
Ion Selectivity and Substrate Specificity" The Structural B asis of Porin Function J.P. R O S E N B U S C H Department of Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, Basel, Switzerland
9 1996 Elsevier Science B. V. All rights reserved
Handbook of Biological Physics Volume 2, edited by W.N. Konings, H.R. Kaback and J.S. Lolkema
599
Contents
1.
Introduction
2.
Overall folding pattern and protein sequences . . . . . . . . . . . . . . . . . . . . . . . . . . .
............................................
3.
The architecture of porin molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
605
4.
Functional properties of porins
609
5.
Aqua incognita: hydration and solvation
6.
Porins as models for other channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
613
Acknowledgements
613
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
601 601
612
613
600
1. Introduction Porins are proteins that form water-filled channels across outer membranes of Gram-negative bacteria. These porins are unusually stable, a significant factor in their early crystallization [ 1]. Porins solved to high resolution (1.8-3.1 A) include two examples from photosynthetic bacteria [2,3], and three from Escherichia coli [4,5]. All of them are homotrimers, although this does not necessarily pertain to all other proteins of the bacterial outer membrane [6]. Some members of the porin family reveal high sequence homology [7], while others do not: the porin from Rhodobacter capsulatus [8], as well as maltoporin from E. coli [9] do not exhibit significant homologies between themselves, nor with the porins belonging to a group with high sequence conservation (60-70%). The latter include matrix porin (the product of the ompF gene [ 10]), osmoporin and phosphoporin (the products of the ompC and phoE genes, respectively [7]. Nonetheless, their architecture is similar overall [ 11 ], a finding which is surprising also in view of their rather distinct function. For the present consideration, which is concerned with the structural basis of the functional characteristics of porins, we focus on two examples that were investigated in Basel for over twenty years: matrix porin [12], an apparently nonspecific pore that appears to allow facilitated diffusion of small (<600 Da), polar molecules (for a review, see Ref. [13]), and maltoporin [14], the function and topology of which has been studied extensively [ 15]. In addition to its general pore function, the former is voltage-gated [16], a property it shares with homologous porins. Its channels are moderately cation-selective, while phosphoporin has a preference for anions [ 17], such as phosphates. Maltoporin fulfils its porin function by allowing the passage of small nutrients through its molecular sieve [ 18], yet it also facilitates the diffusion of malto-oligosaccharides (maltodextrins) which exceed in size the nominal exclusion limit [ 19]. Matrix porin may thus be taken as a paradigm of an unspecific porin, facilitating diffusion at rates proportional to solute concentration, while the rates of the specific substrates of maltoporin follow saturation kinetics [ 13]. Their respective structures, to be discussed presently, reveal that their properties, and some more subtle characteristics not yet mentioned, can be understood on the basis of the structures, and their modes of action can be simulated and the resulting models subjected to rigorous test by tailor-designed mutants.
2. Overall folding pattern and protein sequences The sequences, shown in Fig. 1 as two-dimensional representations based on the topologies of the respective proteins, reveal that matrix porin (panel A) and maltoporin (panel B) exhibit similar arrays of ~-strands that cross the hydrophobic core 601
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of the membrane by 8-9 residues (squares), with aromatic rings demarcating the membrane boundaries. These arrangements should be imagined as folding into a barrel whose sheets are oriented perpendicular to the plane of the membrane. Individual strands are tilted by 40 ~ within the sheet. The side-chains of every other residue in the transmembrane zone are exposedto a medium with a low dielectric constant (bold squares) and are themselves hydrophobic: most of the residues involved are also devoid of a hydrogen bonding potential. Hydrogen bond donors and acceptors in the backbones are saturated by bonds to the corresponding groups in both neighbouring strands, similar as it is found in silk (in a planar array). The intervening residues (light squares) are oriented towards the inside of the barrel where they either contribute to the channel wall, and hence interact with water, or where van der Waals contacts exist with residues present within the channel lumen. The intervening residues may thus be either hydrophilic or hydrophobic, further reducing the constraints for search algorithms of folding patterns. Similarly, the frame may be displaced by so-called 13-bulges (residues 2; 81-82; 290-291 in panel A; 130-131 in panel B), causing further impediment for simple structure predictions. The individual [~-strands are linked by short turns (T) at the periplasmic end (bottom of the panels), and longer loops (L) which are exposed at the cell surface and vary somewhat in length. Loops 3 in both porins bend into the channel and contribute critically to the constriction of the lumen. By this bending, a gap is formed in the barrel wall which is filled in both porins by loops 2 (#L2) from a neighbouring subunit which latches into the notch.
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Structural basis of porin function
605
Apart from these similarities, distinct differences exist between the two molecules. There are 18 transmembrane strands in maltoporin (panel B), as compared to 16 in matrix porin (panel A). In porin, the carboxyterminal end, located on strand 1316, forms a salt-bridge to the amino-terminus on the first half-strand 131", thus forming a quasi-closed barrel. In maltoporin, the amino- and the carboxytermini are also in proximity, but on strands which are adjacent to each other. In the latter protein, a disulfide bridge exists at the exterior surface of the cell which links residues Cys22 and Cys38 in L1. A peculiarity of matrix porin is that in the loops, several short peptide segments exhibit limited periodic structures. Thus, m-helical conformations (rectangles) exist in L3 and L5, while two peptide segments occur in a short 13-structure in L6. Despite these differences, the similarities in both porins suggest divergent evolution, albeit rather distant. The protein sequences in Fig. 2 show the parts of the two sequences which are superimposable on the basis of their tertiary structure. Fifteen strands (131-15) show analogous segments, but interstrand lengths (small figures between 13-strands) often deviate significantly. A number of prediction algorithms have been devised [20-22] but are not immediately relevant in the present context. 3. The architecture of porin molecules The space-filling models of matrix porin (Fig. 3, panel a) and maltoporin (panel b) are based on their X-ray structures and exhibit once more striking resemblance, even though the differences are obvious. Both molecules are encircled by hydrophobic belts in areas where contacts to the membrane core are close. These zones are approximately 25 ]k in width, and aromatic bands demarcate the lipid-water interphase (for better visualization, the carbon atoms in aromatic residues are shown in white). The domains in the periplasmic space are polar and small, reflecting short turns. The extracellular domains in both porins are larger and more polar. Some anionic groups may be linked to phospholipids or lipopolysaccharides (glycolipids) by bridges formed by divalent cations. Positive charges may be linked directly by salt-bridges to the negative charges of the lipopolysaccharides, the lipid moiety of which constitutes the outer leaflet of the bilayer. Outside of the membrane core, and located on [3-strands in register with residues that are exposed to lipids within membrane boundaries, are ionizable; they occur rather frequently. Typical examples are the three lysyl residues which exist next to each other outside the membrane boundary, seen in the I]13 strand of matrix porin (Fig. 1A). In the crevices between subunits, several aromatic residues are visible, particularly in matrix porin. They contribute, in part, to the multiple hydrophobic interactions between neighbouring subunits, which, in the center of the trimers, form a globular, closely packed protein core with a mass of about 15 kDa. These interactions contribute significantly to the stability of porins. Figure 4 shows a schematic representation of a single monomer of maltoporin. Rather unexpectedly, it reveals three residues (N228/D274/Y288) that interact directly with the acyl chains of the lipid (encircled in the figure). The hydrogen bonding potential of their side-chains is saturated by mutual H-bonds. This arrangement is similar to the H-bonding in a periodically arrayed backbone and appears
606
J.P. Rosenbusch
Fig. 3. Space filling models of matrix porin (a) and maltoporin (b) trimers.
Structural basis of porin function
607
r
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Fig. 4. Ribbon representation of a maltoporin monomer. hydrophobic to its surroundings [23]. The representation in Fig. 5a shows the comparison of sections of matrix porin (red) and maltoporin (black). The close similarity of the outline of the [3-barrels is once more remarkable. Due to two additional ]3-strands in each maltoporin monomer, an extension of the elliptical shape of matrix porin to the kidney-bean shape in maltoporin is notable (marked with an asterisk in the topmost subunit in the figure). The sidechains of the three subunits interact closely around the molecular 3-fold axis (triangle) of the trimer. This, as well as the contacts between subunits that are more remote from the symmetry axis also contribute to the protein's stability. In each subunit, the constricting loop (L3) near the central plane of the membrane compartmentalize the barrel into two sections: the water-filled channel proper (open diamond), and the compartment between L3 and the barrel wall (around the arrow), filled with side-chains. The tips of loop 3 are nearly superimposed in matrix porin and in maltoporin, despite the fact that the latter contains 18 residues, while the former consists of 33. In matrix porin, L3 contains an cx-helical segment which closely follows the barrel wall. In either case, the space between L3 and the wall is filled by residues which form both hydrophobic contacts as well as hydrogen bonds (not drawn). The tips of L3 (to the right of the arrow) are in close contact with the barrel walls. An interesting feature exists in matrix porin near the tip of L3 (at the position of the arrow): a hydroxyl group of a serine initiates a series of hydrogen bonds by linkage to two consecutive carboxylate groups (S272-E296-D312). D312 is in turn hydrogen-bonded to two main-chain amide groups in L3 (El 17, F118). The solid sphere indicates the position of the tip (#D74) of the latch in #L2 from the neighbouring subunit, which forms a salt-bridge to the cationic cluster on the barrel wall. The stereo-representation in Fig. 5b illustrates many of the observations outlined so far. It also reveals (though it is difficult to see) that in maltoporin, three
608
J.P. Rosenbusch
Fig. 5. Structural comparison of matrix porin (red) and maltoporin (black). Panel (a): a section across the trimers approximately in the center of the membrane. Panel (b): Stereo-view of the two superimposed monomers. (Courtesy T. Schirmer, unpublished.)
Structural basis of porin function
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loops, in addition to L3, also contribute to the channel constriction: L 1 and L6 from the same subunit, and the latch (#L2) from a neighbouring subunit. The area in the constriction site, with its diameter of 5x5 A, is thus much smaller compared to that in matrix porin (7• This accounts largely for the five-fold reduced conductance of maltoporin, of 0.15 nS as compared to 0.85 nS in matrix porin.
4. Functional properties of porins Can the functional characteristic of the two types of channels, slightly ion-selective but non-specific (matrix porin), and rather specific for malto-oligosaccharides (maltoporin) now be explained on the basis of their structure? The dimensions of the constriction in matrix porin (Fig. 6a) appear large enough to allow unhindered passage of ions (flux about 108 ions/sec/channel), comparable to that observed in gramicidin [24]. The rate of glucose permeation (50 molecules/channel/sec; see [ 16]) though obviously much smaller due to its modest dipole moment [25], appears hardly retarded relative to the diffusion coefficient of this sugar in bulk solvent. This indicates that collisions with the channel wall, and exchange of water of hydration are rare events. In this regard, it is interesting to observe that the strong electrostatic field, originating primarily from the charge segregation in the constriction (the cationic cluster on the channel wall and the carboxylate groups on the constriction loop, see Fig. 6a) actually continues in screw-shape all along the channel [26]. It seems also clear that hydrophobic solutes, with ordered water clathrates covering the hydrophobic surfaces, are considerably retarded [13]. Mutants are available either from nutrient selection [27], or from colicin N resistance [28], and a series of site-directed mutants have been constructed [29]. Their properties are in agreement with expectation with regard to single channel conductance, critical transmembrane voltage closing channel closure, as well as the changed ion selectivities [30,31]. In an evolutionary variant, phospohoporin from E. coli, a single lysyl side-chain that protrudes into the channel and near the center of the constriction site [4] reverses the ion selectivity to a preference for anions [32]. At least two questions remain: does the conformation, as it is present in the crystal and as it is shown in the figure, correspond to the open state? Although intuitively seemingly obvious, it is, in the absence of another crystal form that may be identified as closed state, not possible to answer this question unequivocally. Experiments measuring the water flux, normalized to that of water permeation across pure lipid bilayers, indicates that flux across closed channels (monitored by voltage clamping) is not significantly reduced [33]. The second, and related question concerns the mechanism of channel closing. Does the constricting loop L3 move? Does a change in water structure occur? Are there minor changes in the positions of ionized groups? The channel constriction in maltoporin (Fig. 6b) appears much narrower (5x5 /k) and more crowded by side-chain residues than in the corresponding positions of matrix porin. There exists a segregation of aromatic residues (shown in gray), while the ionizable groups do not show the pattern observed in matrix porin. One tyrosyl residue is found opposite to the aromatic cluster. A longitudinal cross-section of
610
J.P. Rosenbusch
(a)
K16 R82
R13 2 q
(b)
D116 Yl18
W420
Y6
RI09 R82
E43
Y41
Fig. 6. Magnification of the constriction sites of matrix porin (a) and maltoporin (b).
Structural basis of porin function
611
Fig. 7. The channel constriction in maltoporin. Panel (a): Section perpendicular to the membrane plane. The residues of the greasy slide are indicated by their number in the sequence. The space-filling model of maltotriose is positioned according to the electron density. Panel (b): Two ionic tracks (left and right) and the 'greasy slide' (background) surround two maltose molecules (stick model). maltoporin (Fig. 7a) resolves the arrangement of the aromatic residues. The barrel (white lines) is represented with the parts occluding the view of the channel (removed for clarity). The aromatic residues appear arranged in a helical pathway across the narrows of the channel, with a curvature comparable to that of maltodextrin in solution [34]. The contiguous array of aromatic rings thus forms a 'greasy slide'. The tryptophanyl residue (#W74) at the top of the slide is contributed by #L2 of the neighbouring subunit, which latches into the gap left by the bending of L3 into the channel. Two tyr (Y6, Y41) and two trp residues (W420, W358) belong to the pathway, while contacts with phenylalanine F228 have not been observed. Tyrosine 118 (not shown) is on the (white) loop 3, approaching the ligand closely. Soaking experiments with maltotriose have yielded a density in close contact with the three aromatic residues, Y6, Y41, and W420. The ligand is represented here as space filling model to show that its hydrophobic face (yellow, on the left of the model) is in van der Waals distance from the hydrophobic residues. We therefore consider the greasy slide as providing a mechanism that could contribute to the rate enhancement (kcat) of the translocation. In addition to the slide, there exist other interactions with the sugars. Fig. 7b shows two tracks of ionizable residues (R8, D116, R33, H113, and R82, E43, and R109) on both sides of the oligosaccharide. In the background (in blue), the greasy slide can be seen (#W74, Y6, Y41, W420). The ionizable amino acid side-chain residues appear in hydrogen-bonding distance with all sugar hydroxyl groups (here shown are the positions of two maltose residues (yellow/red) from soaking experiments of crystals [35]). These bonds (white lines) are likely to determine both affinity and specificity of the channel for any given substrate as follows. If a solute approaches the channel constriction, it may interact with the first residue(s) of the greasy slide, which guides it into the
612
J.P. Rosenbusch
channel, provided it exhibits a hydrophobic face. The hydrogen bonds formed determine its affinity at the entrance of the channel narrows (KM). If it moves along the slide, the rate is determined by the interaction with the aromatic residues on the one side, and by hydrogen bondings to the ionizable residues in two tracks on the other. In Fig. 7b, two maltose molecules are shown which, according to the electron density map, overlap slightly. The occupancy of each site (#W74, Y6, and Y41, W420) is about 50%. It can also be seen that the binding of the glucose units is slightly off-register relative to the aromatic rings in the greasy slide. It thus appears that the positioning of the molecules is determined by the ionized groups rather than by the stacking of glycosyl rings on aromatic residues. The crystal structure of maltoporin-ligand complexes do provide explanations why certain sugars, such as sucrose, are bound but not translocated [36], but the contributions to the rate enhancement of sugar translocation is not clear in quantitative terms. On the whole, the analogy of the translocator to an enzyme is suggestive, and it will be interesting to see to what extent the concepts described here apply to more specific permeases. But detailed mechanistic questions remain unresolved. The formation of a hydrogen bonding networks appears to explain specificity. How is this related, in detail, to the observed rates of translocation? Are there conformational changes in the side-chain residues involved directly in translocation? Or does a concerted motion effectuate translocation? Can the diffusion be conceived of as one-dimensional? From the crystallographic data, all that is clear is that the ]3-barrels are not distinguishable in the liganded and the unliganded states. Changes in the conformation of the side-chain residues were not observed [35], but higher resolution will required for definitive conclusions.
5. Aqua incognita: hydration and solvation The topology of maltoporin channels reveals unequivocally that there is no space for water molecules in the channel if it is occupied by specific ligands. The situation in matrix porin is drastically different. There clearly exists space for a large number of water molecules, of which some have been found ordered in crystallographic analyses [37,38]. Yet nothing is known about the hydration of the various ionizable residues. Clearly, the pK-values of the three arginyl residues in the cationic cluster cannot correspond to the values found in bulk solution. Calculations have revealed, for instance, that the in situ pK value of R82 is close to neutral [26]. The guanidinium groups are in close (n-re) contact, however, so that caution must rule when interpreting such results. Similar calculations have revealed that the buried carboxyl groups between L3 and the barrel wall are uncharged. Again this appears intuitively attractive, but we know as yet little about the local effect of an external electric field on the state of ionization of these residues. This is clearly a significant problem when the question is raised whether the constriction loop L3 moves during channels closure [39]. Another question will require further investigation: so far, it has been tacitly assumed that no shielding of fixed charges occur by mobile ions. This could not have been observed in the X-ray analysis, due to the poor scattering of counter-ions such as sodium or potassium. It also depends on the residence times
Structural basis of porin function
613
of such ions in the vicinity of their counterions. The use of other ions, such as Cs +, Ba +2, or Tb +3 may help to resolve these questions. Terbium may be followed also by fluorescence, and may thus open different time windows. 6. Porins as models for other channels
The detailed results from the structural and the functional studies, given in outline above, illustrate that the results from high resolution structural studies and single channel recordings of channel-forming proteins, allow solute translocation to be understood at a molecular level. The contributions of molecular pathology, be that by the application of strong selective pressures (growth on carbon sources which, due to the constriction size, are not normally taken up by E. coli [27], or by the exposure to bacterial toxins [28]) has clearly proven valuable. Mutations that are tailor-designed [29] on the basis of structural information at atomic resolution may be more intriguing, as problems can be addressed much more specifically. Answering questions concerning the trajectories of solutes requires further simulations of the electrostatic properties of porin, the prediction of the effects of changes in charge distributions, and detailed molecular dynamics simulations [39]. The manoeuvrability of bacteria such as E. coli has the obvious advantage that the models proposed by computational approaches can be subjected to rigorous testing. In addition, mapping the topology of the surface by means of atomic force microscopy allows protein-lipid interactions to be studied in detail [40] and may give information on conformational changes in the loops. Interatomic distance measurements by solid-state NMR spectroscopy may, moreover, prove a very powerful tool in assessing channel dynamics, conformational changes, and solute flux. Matrix porin may also be viewed as a model for other channels. Nanotubes have recently been studied by allowing self-assembly of small synthetic peptides [41]. In view of the fact that much attention has been focused on mimicking biological function, it may be intriguing to consider porin as natural nanotubes which, moreover, are voltage-gated (a poristcr?). These proteins can be prepared in large quantities, and the genetics makes them versatile, with changes engineered at specific sites, and the results accessible to analysis by crystallographic methods. It will be a challenging task for the future to determine to what extent such ideas may be realistic. Acknowledgments Supported by grants of the Swiss National Science Foundation. I thank Lucienne Letellier for allowing me to peruse her contribution to this volume in advance. The critical reading of this manuscript by Tilman Schirmcr, and his providing me generously with illustrations is highly appreciated. The constructive comments, and the criticism of the manuscript by Dr. Robert S. Eisenberg, Rush University, Chicago IL, are most gratefully acknowledged. References I. 2.
Garavito, R.M. and Rosenbusch, J.P. (1980) J. Cell. Biol. 86, 327-329. Weiss, M.S., Abele, U., Weckesser, J., Welte, W., Schiltz, E. and Schulz, G.E. (1991) Science 254,
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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
J.P. Rosenbusch
1627-1630. Kreusch, A., Neubtiser, E., Schiltz, E., Weckesser, J. and Schulz, G.E. (1994) Protein Sci. 3, 58-63. Cowan, S.W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R.A., Jansonius, J.N. and Rosenbusch, J.P. (1992) Nature 358, 727-733. Schirmer, T., Keller, T.A., Wang, Y.-F. and Rosenbusch, J.P. (1995) Science 264, 914-916. Letellier, L. and Bonhivers, M. (1996) in: Transport Processes in Membranes, eds W. Konings, H.R. Kaback and J.S. Lolkema. Handbook of Biological Physics, Vol. 2, pp. 615-636. Elsevier, Amsterdam. Mizuno, T., Chou, M.-Y. and Inouye, M. (1983) J. Biol. Chem. 258, 6932-6940. Schiltz, E., Kreusch, A., Nestel, U. and Schulz, G.E. (1991) Eur. J. Biochem. 199, 587-594. C16ment, J.M. and Hofnung, M. (1981). Cell 27, 507-514. Chen, R., Krfimer, C., Schidmayr, W., Chen-Schmeisser, U. and Henning, U. (1982) Biochem. J. 203, 33-43. Pauptit, R.A., Schirmer, T., Jansonius, J.N., Rosenbusch, J.P., Parker, M.W., Tucker, A.D., Tsernoglou, D., Weiss, M.S. and Schulz, G.E. (1991) J. Struct. Biol. 107, 136-145. Rosenbusch, J.P. (1974) J. Biol. Chem. 249, 8019-8029. Nikaido, H. (1992) Mol. Microbiol. 6, 435-442. Neuhaus, J.-M., Schindler, H. and Rosenbusch, J.P. (1983) EMBO J. 2, 1987-191 Charbit, A., Gehring, K., Nikaido, H., Ferenci, T. and Hofnung, M. (1988) J. Mol. Biol. 201,487-496. Schindler, H. and Rosenbusch, J.P. (1978) Proc. Natl. Acad. Sci. USA 75, 3751-3755. Benz, R., Schmid, A. and Hancock, R.E.W. (1985) J. Bacteriol. 162, 722-727. von Meyenburg, K. and Nikaido, H. (1977) Biochem. Biophys. Res. Commun. 78, 1100-1107. Wandersman, C., Schwartz, M. and Ferenci, T. (1979) J. Bacteriol. 140, 1-13. Paul, C. and Rosenbusch, J.P. (1985) EMBO J. 4, 1593-1597. Jeanteur, D., Lakey, J.H. and Pattus, F. (1991) Mol. Microbiol. 5, 2153-2164 Schirmer, T. and Cowan, S.W. (1993) Prot. Sci. 2, 1361-1363. Rosenbusch, J.P. (1990) Experientia 46, 167-173. Anderson, O. (1983) Biophys. J. 53, 119-133. Tait•M.J.•Suggett•A.•Franks•F.•Ab•ett•S.andQuickenden•P.A.(•972)J.S••uti•nChem.•• 131-151. Karshikoff, A., Spassov, V., Cowan, S.A., Ladenstein, R. and Schirmer, T. (1994) J. Mol. Biol. 240, 372-384. Benson, S.A., Occi, J.L.L. and Sampson, B.A. (1988) J. Mol. Biol. 203, 961-970. Jeanteur, D., Schirmer, T., Fourel, D., Simonet, V., Rummel, G., Widmer, C., Rosenbusch, J.P., Pattus, F. and Pages, J.-M. (1994) Proc. Natl. Acad. Sci. USA 91, 10675-10679. Prilipov, A. and Rosenbusch, J.P. (1996) In preparation. Saint, N., Widmer, C., Prilipov, A., Luckey, M., Lou, K.-L., Schirmer, T. and Rosenbusch, J.P. (1996) J. Biol. Chem., in press. Lou, K.-L., Saint, N., Rummel, G., Benson, S.A., Rosenbusch, J.P. and Schirmer, T. (1996) J. Biol. Chem., in press. Bauer, K., Struyve, M., Bosch, D., Benz, R. and Tommassen, J. (1989) J. Biol. Chem. 264, 16393-16398. Steiert, M. (1993). Structure and function of phosphoporin: Properties of a porin from the outer membrane of Escherichia coli. Ph.D. Thesis (University of Basel). Goldsmith, E., Sprang, S. and Flettrick, R. (1982) J. Mol. Biol. 156, 411--427. Dutzler, R., Wang, Y.-F., Rosenbusch, J.P. and Schirmer, T. (1996) Structure 4, 127-134. Andersen, C., Jordy, M. and Benz, R. (1995) J. Gen. Physiol. 105, 385-401. Weiss, M.S. and Schulz, G.E. (1992) J. Mol. Biol. 227, 493-509. Cowan, S.W. and Schirmer, T. (1996) Personal communication. Watanabe, M., Rosenbusch, J.P., Schirmer, T. and Karplus, M. (1996) Computer simulations of the OmpF porin from the outer membrane from Escherichia coli. Biophys. J., in press. Schabert, F.A., Henn, C. and Engel, A. (1995) Science 268, 92-94. Ghadiri, M.R., Granja, J.R. and Buehler, L.K. (1994) Nature 369, 301-304.
Ion Selectivity and Substrate Specificity" The Structural B asis of Porin Function J.P. R O S E N B U S C H Department of Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, Basel, Switzerland
9 1996 Elsevier Science B. V. All rights reserved
Handbook of Biological Physics Volume 2, edited by W.N. Konings, H.R. Kaback and J.S. Lolkema
599
Contents
1.
Introduction
2.
Overall folding pattern and protein sequences . . . . . . . . . . . . . . . . . . . . . . . . . . .
............................................
3.
The architecture of porin molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
605
4.
Functional properties of porins
609
5.
Aqua incognita: hydration and solvation
6.
Porins as models for other channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
613
Acknowledgements
613
.................................. .............................
........................................
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
601 601
612
613
600
1. Introduction Porins are proteins that form water-filled channels across outer membranes of Gram-negative bacteria. These porins are unusually stable, a significant factor in their early crystallization [ 1]. Porins solved to high resolution (1.8-3.1 A) include two examples from photosynthetic bacteria [2,3], and three from Escherichia coli [4,5]. All of them are homotrimers, although this does not necessarily pertain to all other proteins of the bacterial outer membrane [6]. Some members of the porin family reveal high sequence homology [7], while others do not: the porin from Rhodobacter capsulatus [8], as well as maltoporin from E. coli [9] do not exhibit significant homologies between themselves, nor with the porins belonging to a group with high sequence conservation (60-70%). The latter include matrix porin (the product of the ompF gene [ 10]), osmoporin and phosphoporin (the products of the ompC and phoE genes, respectively [7]. Nonetheless, their architecture is similar overall [ 11 ], a finding which is surprising also in view of their rather distinct function. For the present consideration, which is concerned with the structural basis of the functional characteristics of porins, we focus on two examples that were investigated in Basel for over twenty years: matrix porin [12], an apparently nonspecific pore that appears to allow facilitated diffusion of small (<600 Da), polar molecules (for a review, see Ref. [13]), and maltoporin [14], the function and topology of which has been studied extensively [ 15]. In addition to its general pore function, the former is voltage-gated [16], a property it shares with homologous porins. Its channels are moderately cation-selective, while phosphoporin has a preference for anions [ 17], such as phosphates. Maltoporin fulfils its porin function by allowing the passage of small nutrients through its molecular sieve [ 18], yet it also facilitates the diffusion of malto-oligosaccharides (maltodextrins) which exceed in size the nominal exclusion limit [ 19]. Matrix porin may thus be taken as a paradigm of an unspecific porin, facilitating diffusion at rates proportional to solute concentration, while the rates of the specific substrates of maltoporin follow saturation kinetics [ 13]. Their respective structures, to be discussed presently, reveal that their properties, and some more subtle characteristics not yet mentioned, can be understood on the basis of the structures, and their modes of action can be simulated and the resulting models subjected to rigorous test by tailor-designed mutants.
2. Overall folding pattern and protein sequences The sequences, shown in Fig. 1 as two-dimensional representations based on the topologies of the respective proteins, reveal that matrix porin (panel A) and maltoporin (panel B) exhibit similar arrays of ~-strands that cross the hydrophobic core 601
602
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J.P. Rosenbusch
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Structural basis of porin function
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J.P. Rosenbusch
of the membrane by 8-9 residues (squares), with aromatic rings demarcating the membrane boundaries. These arrangements should be imagined as folding into a barrel whose sheets are oriented perpendicular to the plane of the membrane. Individual strands are tilted by 40 ~ within the sheet. The side-chains of every other residue in the transmembrane zone are exposedto a medium with a low dielectric constant (bold squares) and are themselves hydrophobic: most of the residues involved are also devoid of a hydrogen bonding potential. Hydrogen bond donors and acceptors in the backbones are saturated by bonds to the corresponding groups in both neighbouring strands, similar as it is found in silk (in a planar array). The intervening residues (light squares) are oriented towards the inside of the barrel where they either contribute to the channel wall, and hence interact with water, or where van der Waals contacts exist with residues present within the channel lumen. The intervening residues may thus be either hydrophilic or hydrophobic, further reducing the constraints for search algorithms of folding patterns. Similarly, the frame may be displaced by so-called 13-bulges (residues 2; 81-82; 290-291 in panel A; 130-131 in panel B), causing further impediment for simple structure predictions. The individual [~-strands are linked by short turns (T) at the periplasmic end (bottom of the panels), and longer loops (L) which are exposed at the cell surface and vary somewhat in length. Loops 3 in both porins bend into the channel and contribute critically to the constriction of the lumen. By this bending, a gap is formed in the barrel wall which is filled in both porins by loops 2 (#L2) from a neighbouring subunit which latches into the notch.
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Fig. 2. Sequencecomparisonof maltoporin(LamB)and matrixporin (OmpF). (CourtesyT. Schirmer, unpublished.)
Structural basis of porin function
605
Apart from these similarities, distinct differences exist between the two molecules. There are 18 transmembrane strands in maltoporin (panel B), as compared to 16 in matrix porin (panel A). In porin, the carboxyterminal end, located on strand 1316, forms a salt-bridge to the amino-terminus on the first half-strand 131", thus forming a quasi-closed barrel. In maltoporin, the amino- and the carboxytermini are also in proximity, but on strands which are adjacent to each other. In the latter protein, a disulfide bridge exists at the exterior surface of the cell which links residues Cys22 and Cys38 in L1. A peculiarity of matrix porin is that in the loops, several short peptide segments exhibit limited periodic structures. Thus, m-helical conformations (rectangles) exist in L3 and L5, while two peptide segments occur in a short 13-structure in L6. Despite these differences, the similarities in both porins suggest divergent evolution, albeit rather distant. The protein sequences in Fig. 2 show the parts of the two sequences which are superimposable on the basis of their tertiary structure. Fifteen strands (131-15) show analogous segments, but interstrand lengths (small figures between 13-strands) often deviate significantly. A number of prediction algorithms have been devised [20-22] but are not immediately relevant in the present context. 3. The architecture of porin molecules The space-filling models of matrix porin (Fig. 3, panel a) and maltoporin (panel b) are based on their X-ray structures and exhibit once more striking resemblance, even though the differences are obvious. Both molecules are encircled by hydrophobic belts in areas where contacts to the membrane core are close. These zones are approximately 25 ]k in width, and aromatic bands demarcate the lipid-water interphase (for better visualization, the carbon atoms in aromatic residues are shown in white). The domains in the periplasmic space are polar and small, reflecting short turns. The extracellular domains in both porins are larger and more polar. Some anionic groups may be linked to phospholipids or lipopolysaccharides (glycolipids) by bridges formed by divalent cations. Positive charges may be linked directly by salt-bridges to the negative charges of the lipopolysaccharides, the lipid moiety of which constitutes the outer leaflet of the bilayer. Outside of the membrane core, and located on [3-strands in register with residues that are exposed to lipids within membrane boundaries, are ionizable; they occur rather frequently. Typical examples are the three lysyl residues which exist next to each other outside the membrane boundary, seen in the I]13 strand of matrix porin (Fig. 1A). In the crevices between subunits, several aromatic residues are visible, particularly in matrix porin. They contribute, in part, to the multiple hydrophobic interactions between neighbouring subunits, which, in the center of the trimers, form a globular, closely packed protein core with a mass of about 15 kDa. These interactions contribute significantly to the stability of porins. Figure 4 shows a schematic representation of a single monomer of maltoporin. Rather unexpectedly, it reveals three residues (N228/D274/Y288) that interact directly with the acyl chains of the lipid (encircled in the figure). The hydrogen bonding potential of their side-chains is saturated by mutual H-bonds. This arrangement is similar to the H-bonding in a periodically arrayed backbone and appears
606
J.P. Rosenbusch
Fig. 3. Space filling models of matrix porin (a) and maltoporin (b) trimers.
Structural basis of porin function
607
r
)
Fig. 4. Ribbon representation of a maltoporin monomer. hydrophobic to its surroundings [23]. The representation in Fig. 5a shows the comparison of sections of matrix porin (red) and maltoporin (black). The close similarity of the outline of the [3-barrels is once more remarkable. Due to two additional ]3-strands in each maltoporin monomer, an extension of the elliptical shape of matrix porin to the kidney-bean shape in maltoporin is notable (marked with an asterisk in the topmost subunit in the figure). The sidechains of the three subunits interact closely around the molecular 3-fold axis (triangle) of the trimer. This, as well as the contacts between subunits that are more remote from the symmetry axis also contribute to the protein's stability. In each subunit, the constricting loop (L3) near the central plane of the membrane compartmentalize the barrel into two sections: the water-filled channel proper (open diamond), and the compartment between L3 and the barrel wall (around the arrow), filled with side-chains. The tips of loop 3 are nearly superimposed in matrix porin and in maltoporin, despite the fact that the latter contains 18 residues, while the former consists of 33. In matrix porin, L3 contains an cx-helical segment which closely follows the barrel wall. In either case, the space between L3 and the wall is filled by residues which form both hydrophobic contacts as well as hydrogen bonds (not drawn). The tips of L3 (to the right of the arrow) are in close contact with the barrel walls. An interesting feature exists in matrix porin near the tip of L3 (at the position of the arrow): a hydroxyl group of a serine initiates a series of hydrogen bonds by linkage to two consecutive carboxylate groups (S272-E296-D312). D312 is in turn hydrogen-bonded to two main-chain amide groups in L3 (El 17, F118). The solid sphere indicates the position of the tip (#D74) of the latch in #L2 from the neighbouring subunit, which forms a salt-bridge to the cationic cluster on the barrel wall. The stereo-representation in Fig. 5b illustrates many of the observations outlined so far. It also reveals (though it is difficult to see) that in maltoporin, three
608
J.P. Rosenbusch
Fig. 5. Structural comparison of matrix porin (red) and maltoporin (black). Panel (a): a section across the trimers approximately in the center of the membrane. Panel (b): Stereo-view of the two superimposed monomers. (Courtesy T. Schirmer, unpublished.)
Structural basis of porin function
609
loops, in addition to L3, also contribute to the channel constriction: L 1 and L6 from the same subunit, and the latch (#L2) from a neighbouring subunit. The area in the constriction site, with its diameter of 5x5 A, is thus much smaller compared to that in matrix porin (7• This accounts largely for the five-fold reduced conductance of maltoporin, of 0.15 nS as compared to 0.85 nS in matrix porin.
4. Functional properties of porins Can the functional characteristic of the two types of channels, slightly ion-selective but non-specific (matrix porin), and rather specific for malto-oligosaccharides (maltoporin) now be explained on the basis of their structure? The dimensions of the constriction in matrix porin (Fig. 6a) appear large enough to allow unhindered passage of ions (flux about 108 ions/sec/channel), comparable to that observed in gramicidin [24]. The rate of glucose permeation (50 molecules/channel/sec; see [ 16]) though obviously much smaller due to its modest dipole moment [25], appears hardly retarded relative to the diffusion coefficient of this sugar in bulk solvent. This indicates that collisions with the channel wall, and exchange of water of hydration are rare events. In this regard, it is interesting to observe that the strong electrostatic field, originating primarily from the charge segregation in the constriction (the cationic cluster on the channel wall and the carboxylate groups on the constriction loop, see Fig. 6a) actually continues in screw-shape all along the channel [26]. It seems also clear that hydrophobic solutes, with ordered water clathrates covering the hydrophobic surfaces, are considerably retarded [13]. Mutants are available either from nutrient selection [27], or from colicin N resistance [28], and a series of site-directed mutants have been constructed [29]. Their properties are in agreement with expectation with regard to single channel conductance, critical transmembrane voltage closing channel closure, as well as the changed ion selectivities [30,31]. In an evolutionary variant, phospohoporin from E. coli, a single lysyl side-chain that protrudes into the channel and near the center of the constriction site [4] reverses the ion selectivity to a preference for anions [32]. At least two questions remain: does the conformation, as it is present in the crystal and as it is shown in the figure, correspond to the open state? Although intuitively seemingly obvious, it is, in the absence of another crystal form that may be identified as closed state, not possible to answer this question unequivocally. Experiments measuring the water flux, normalized to that of water permeation across pure lipid bilayers, indicates that flux across closed channels (monitored by voltage clamping) is not significantly reduced [33]. The second, and related question concerns the mechanism of channel closing. Does the constricting loop L3 move? Does a change in water structure occur? Are there minor changes in the positions of ionized groups? The channel constriction in maltoporin (Fig. 6b) appears much narrower (5x5 /k) and more crowded by side-chain residues than in the corresponding positions of matrix porin. There exists a segregation of aromatic residues (shown in gray), while the ionizable groups do not show the pattern observed in matrix porin. One tyrosyl residue is found opposite to the aromatic cluster. A longitudinal cross-section of
610
J.P. Rosenbusch
(a)
K16 R82
R13 2 q
(b)
D116 Yl18
W420
Y6
RI09 R82
E43
Y41
Fig. 6. Magnification of the constriction sites of matrix porin (a) and maltoporin (b).
Structural basis of porin function
611
Fig. 7. The channel constriction in maltoporin. Panel (a): Section perpendicular to the membrane plane. The residues of the greasy slide are indicated by their number in the sequence. The space-filling model of maltotriose is positioned according to the electron density. Panel (b): Two ionic tracks (left and right) and the 'greasy slide' (background) surround two maltose molecules (stick model). maltoporin (Fig. 7a) resolves the arrangement of the aromatic residues. The barrel (white lines) is represented with the parts occluding the view of the channel (removed for clarity). The aromatic residues appear arranged in a helical pathway across the narrows of the channel, with a curvature comparable to that of maltodextrin in solution [34]. The contiguous array of aromatic rings thus forms a 'greasy slide'. The tryptophanyl residue (#W74) at the top of the slide is contributed by #L2 of the neighbouring subunit, which latches into the gap left by the bending of L3 into the channel. Two tyr (Y6, Y41) and two trp residues (W420, W358) belong to the pathway, while contacts with phenylalanine F228 have not been observed. Tyrosine 118 (not shown) is on the (white) loop 3, approaching the ligand closely. Soaking experiments with maltotriose have yielded a density in close contact with the three aromatic residues, Y6, Y41, and W420. The ligand is represented here as space filling model to show that its hydrophobic face (yellow, on the left of the model) is in van der Waals distance from the hydrophobic residues. We therefore consider the greasy slide as providing a mechanism that could contribute to the rate enhancement (kcat) of the translocation. In addition to the slide, there exist other interactions with the sugars. Fig. 7b shows two tracks of ionizable residues (R8, D116, R33, H113, and R82, E43, and R109) on both sides of the oligosaccharide. In the background (in blue), the greasy slide can be seen (#W74, Y6, Y41, W420). The ionizable amino acid side-chain residues appear in hydrogen-bonding distance with all sugar hydroxyl groups (here shown are the positions of two maltose residues (yellow/red) from soaking experiments of crystals [35]). These bonds (white lines) are likely to determine both affinity and specificity of the channel for any given substrate as follows. If a solute approaches the channel constriction, it may interact with the first residue(s) of the greasy slide, which guides it into the
612
J.P. Rosenbusch
channel, provided it exhibits a hydrophobic face. The hydrogen bonds formed determine its affinity at the entrance of the channel narrows (KM). If it moves along the slide, the rate is determined by the interaction with the aromatic residues on the one side, and by hydrogen bondings to the ionizable residues in two tracks on the other. In Fig. 7b, two maltose molecules are shown which, according to the electron density map, overlap slightly. The occupancy of each site (#W74, Y6, and Y41, W420) is about 50%. It can also be seen that the binding of the glucose units is slightly off-register relative to the aromatic rings in the greasy slide. It thus appears that the positioning of the molecules is determined by the ionized groups rather than by the stacking of glycosyl rings on aromatic residues. The crystal structure of maltoporin-ligand complexes do provide explanations why certain sugars, such as sucrose, are bound but not translocated [36], but the contributions to the rate enhancement of sugar translocation is not clear in quantitative terms. On the whole, the analogy of the translocator to an enzyme is suggestive, and it will be interesting to see to what extent the concepts described here apply to more specific permeases. But detailed mechanistic questions remain unresolved. The formation of a hydrogen bonding networks appears to explain specificity. How is this related, in detail, to the observed rates of translocation? Are there conformational changes in the side-chain residues involved directly in translocation? Or does a concerted motion effectuate translocation? Can the diffusion be conceived of as one-dimensional? From the crystallographic data, all that is clear is that the ]3-barrels are not distinguishable in the liganded and the unliganded states. Changes in the conformation of the side-chain residues were not observed [35], but higher resolution will required for definitive conclusions.
5. Aqua incognita: hydration and solvation The topology of maltoporin channels reveals unequivocally that there is no space for water molecules in the channel if it is occupied by specific ligands. The situation in matrix porin is drastically different. There clearly exists space for a large number of water molecules, of which some have been found ordered in crystallographic analyses [37,38]. Yet nothing is known about the hydration of the various ionizable residues. Clearly, the pK-values of the three arginyl residues in the cationic cluster cannot correspond to the values found in bulk solution. Calculations have revealed, for instance, that the in situ pK value of R82 is close to neutral [26]. The guanidinium groups are in close (n-re) contact, however, so that caution must rule when interpreting such results. Similar calculations have revealed that the buried carboxyl groups between L3 and the barrel wall are uncharged. Again this appears intuitively attractive, but we know as yet little about the local effect of an external electric field on the state of ionization of these residues. This is clearly a significant problem when the question is raised whether the constriction loop L3 moves during channels closure [39]. Another question will require further investigation: so far, it has been tacitly assumed that no shielding of fixed charges occur by mobile ions. This could not have been observed in the X-ray analysis, due to the poor scattering of counter-ions such as sodium or potassium. It also depends on the residence times
Structural basis of porin function
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of such ions in the vicinity of their counterions. The use of other ions, such as Cs +, Ba +2, or Tb +3 may help to resolve these questions. Terbium may be followed also by fluorescence, and may thus open different time windows. 6. Porins as models for other channels
The detailed results from the structural and the functional studies, given in outline above, illustrate that the results from high resolution structural studies and single channel recordings of channel-forming proteins, allow solute translocation to be understood at a molecular level. The contributions of molecular pathology, be that by the application of strong selective pressures (growth on carbon sources which, due to the constriction size, are not normally taken up by E. coli [27], or by the exposure to bacterial toxins [28]) has clearly proven valuable. Mutations that are tailor-designed [29] on the basis of structural information at atomic resolution may be more intriguing, as problems can be addressed much more specifically. Answering questions concerning the trajectories of solutes requires further simulations of the electrostatic properties of porin, the prediction of the effects of changes in charge distributions, and detailed molecular dynamics simulations [39]. The manoeuvrability of bacteria such as E. coli has the obvious advantage that the models proposed by computational approaches can be subjected to rigorous testing. In addition, mapping the topology of the surface by means of atomic force microscopy allows protein-lipid interactions to be studied in detail [40] and may give information on conformational changes in the loops. Interatomic distance measurements by solid-state NMR spectroscopy may, moreover, prove a very powerful tool in assessing channel dynamics, conformational changes, and solute flux. Matrix porin may also be viewed as a model for other channels. Nanotubes have recently been studied by allowing self-assembly of small synthetic peptides [41]. In view of the fact that much attention has been focused on mimicking biological function, it may be intriguing to consider porin as natural nanotubes which, moreover, are voltage-gated (a poristcr?). These proteins can be prepared in large quantities, and the genetics makes them versatile, with changes engineered at specific sites, and the results accessible to analysis by crystallographic methods. It will be a challenging task for the future to determine to what extent such ideas may be realistic. Acknowledgments Supported by grants of the Swiss National Science Foundation. I thank Lucienne Letellier for allowing me to peruse her contribution to this volume in advance. The critical reading of this manuscript by Tilman Schirmcr, and his providing me generously with illustrations is highly appreciated. The constructive comments, and the criticism of the manuscript by Dr. Robert S. Eisenberg, Rush University, Chicago IL, are most gratefully acknowledged. References I. 2.
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