Structure and function of the channel

WOLFRAM WELTE, UWE NESTEL, THOMAS WACKER, and KAy DIEDERICHS

Fakultiit fUr Biologie, Universitiit Konstanz, Konstanz, Germany

The outer membrane bilayer of gram-negative bacteria, which except in the case of porin from Rhodobacter capsulatus [22] and encapsulates completely the plasma membrane, seems to act as a porins from Pseudomonas aeruginosa [23]. protection system against noxious macromolecules like proteases and bile-salt micelles [1]. The external monolayer is composed of Classification of porins according to their observed functions lipopolysaccharides with a variable carbohydrat.e composi~i?n. Long oligosaccharide chains protect the cells agamst recogmtlOn The following functional characteristics and classes of porins by the complement system [2, 3] and hydrophobic substances [4]. have emerged from these studies. The other monolayer facing the peri plasmic space is mainly (1) The majority of porins belong to the class of nonspe~ific composed of phospholipid. so-called "general diffusion" porins [24, 25] and have typical This protection system should nevertheless allow free access of exclusion limits of 600 Da, with extremes of 5000 Da. Each small biomolecules like mono- and disaccharides, nucleosides, bacterial cell seems to have one of these porins in high copy and amino acids to the cytoplasmic membrane. The proteins numbers in its outer membrane. They show slightly different allowing for rapid diffusion of these molecules across the outer permeabilities for cations (Pc) and anions (Pa), respectively. membrane are called porins [5]. The "general diffusion porins" Pc/Pa ratios vary typically between 0.3 and 4 [25]. Porins show a form aqueous channels with an exclusion limit of typically 600 Da voltage-induced closing, leading to a macroscopic reduction of and extremes of 5000 Da. Due to their high copy number they current in 1 M KCl by a factor of between 0.85 and 0.15 [7, 14, 16, form the major integral protein component of the outer mem­ 26, 27]. The voltage at which this reduction takes place, the brane and turn it into a molecular sieve. A single cell of E. coli has "gating voltage," seems to depend upon the orientation of the been estimated to possess approximately 105 OmpF porin mole­ external field relative to the porin [28] and values of between 5 cules [6]. m V and 200 m V have been reported. Approximately three Purification, reconstitution into black lipid bilayers and charges move upon gating [28]. electrical measurements A voltage-dependent anion channel protein (VDAC) in the mitochondrial outer membrane [29] may have evolved from these Porins can be isolated and purified by chromatography after procaryotic porins. It has a higher exclusion limit (2500) than most solubilization or preferential extraction of the outer membrane general diffusion porins, shows voltage gating at ~ 30 m V [30] and with detergents, even with sodium dodecylsulfate [6-11]. For does not seem to form trimers [31-34]. For regulatory porposes, most of the preparations a porin trimer has been reported [8, VDAC seems to have evolved the ability to form complexes with 12-14]. The trimers insert spontaneously into black phospholipid hexokinase [35] to regulate uptake of carbohydrate, with creatine membranes of the Montal-Mueller or Mueller-Rudin type. Their kinase [36], with a benzodiazepine receptor [37] to regulate electrical properties can be studied by measurement with elec­ steroidogenesis and with a water-soluble modulating protein [38]. trodes of the current across these porin-doped black lipid bilayer A large-conductance anion channel in astrocytic plasma mem­ membranes [7, 15-18]. The bilayer can be made up from asym­ branes is highly homologous with VDAC [39]. Porins from metric LPS-phospholipid bilayers mimicking the outer membrane chloroplast outer membranes [40] and mycobacteria [41] have [19] . Patch-clamp techniques have also been used [20]. S~~ll also been reported. radio labeled substances have been used to study the permeabIlIty (2) "Specific porins" possess a binding site which is moderately of porins in whole cells and porin containing liposomes. Diffusion specific for a certain class of substances (dissociation constants are rates of permeating solutes can be studied by the swelling typically around Kd ~100 /-Lmol [24]) and are usually expressed behavior of liposomes which are initially prepared in presence of under certain growth limitation conditions at low copy numbers. a nonpermeable polysaccharide. After dilution into isoosmolar They scem to be always part of an energy-driven uptake system solutions of small permeating solutes an increase of turbidity due like a phosphotransferase (PTS) system [42,43] or a ATP binding to the swelling is observed [21]. cassette (ABC) system [44]. Specific porins have been found for Trimers can be dissociated to monomers by heat- and detergent mono- and disaccharides (LamB, ScrY), nucleosides (TsX) [45] treatment [8]. Usually, the monomers are in some respect dena­ and anions (protein P from Pseudomonas aeruginosa). These tured, as reassociation of monomers leads to inactive trimers [8] porins show a lower single channel conductivity than general diffusion porins which can be blocked upon addition of the substrate [46]. The transport rate of specific porins saturates in a Michaelis-Menten like manner when the binding site is occupied.

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Amino acid sequences and conclusions related to them obtained [63, 64]. Garavito et al also obtained crystals of Malto­ An increasing number of porin sequences is known. General porin (LamB) and OmpA from E. coli [65]. The former could be diffusion porins and specific porins typically have masses of 30 to improved by Stauffer et al [66]. Crystals of phosphoporin (PhoE) 40 kD and 30 to 50 kD, respectively. Compared with other integral from E. coli have also been obtained [67]. As the phase determi­ membrane proteins, the amino acid compositions of porin se­ nation by multiple isomorphous replacement (MIR) was delayed, quences are rather polar and lack hydrophobic segments long electron microscopic work using two-dimensional crystalline enough to span membrane bilayers [47]. In accord with their sheets [68] was done in parallel. Engel et al [69] and lap, Walian rather hydrophilic amino acid sequence, porins are exported to and Gehring [70] used image filtering and three-dimensional the periplasmic space as water soluble precursors [48, 49] with a reconstruction techniques of stained and unstained, respectively, signal sequence like other periplasmic proteins. The removal of two dimensional crystals to obtain the structure at a resolution of the signal sequence preceeds trimerization [50]. It is conceivable 28 A a~d 6 A, res\?ectively. The resolution of projection maps was that the removal of the signal sequence allows the formation of a even hIgher (3.2 A) [71]. A large /3-barrel forming the surface of hydrophobic core by N- and C-termini of three porin molecules. the protein in contact with the membrane lipid phase was inferred The energy of formation of this core may stabilize the trimer with from these studies. The appearance of porin trimers in two its membrane exposed hydrophobic surface against the water­ dimensional crystals is so characteristic that in some bacteria the soluble conformation and thus trigger the insertion into the outer existence of porins has been inferred from electron micrographs membrane. Other porins or porin-like molecules like OmpA or of whole cells [72, 73]. VDAC, which have N- or C-terminal extensions, thus should not A moderately cation selective porin has been purified from the form trimers and might be able to exist both in the water-soluble facultatively phototrophic purple bacterium Rhodobacter capsula­ and the membrane bound conformation. tus [74]. The crystals of this porin [75] could be improved to When sequences are compared no global similarity is found, not diffract to 1.8 A resolution [7] and were analyzed with X-ray even within one of the two porin subclasses. Only porins within crystallography [76-79]. one subclass and from phylogenetically related organisms show In the meanwhile, a new trigonal crystal form of OmpF [80] sequence similarities. Several classifications of porins have been allowed the solution of the structure of this E. coli porin and of the proposed. Schiltz et al [51] suggest 10 groups, leanteur, Lakey and related phosphoporin PhoE as well [81]. Another porin from the Pattus [52] propose a phylogenetic tree for porins. As there is no purple bacterium Rhodopseudomonas blastica was crystallized [8] universal sequence classification for porins yet, general phyloge­ and the structure solved to 2 A resolution [82]. The structure netic relations of bacteria should be taken into regard as was done analyses of two specific porins, maltoporin (LamB) [66] and by Nikaido [53]. sucrose porin (ScrY) [83] are underway. Tommassen [54] compared the rather homologous sequences of the three non-specific porins OmpF, OmpC and PhoE from Structure of bacterial porins as determined by X-ray Escherichia coli and found that there are eight zones within which crystallography the sequences are most variable, separated by rather well con­ General description served stretches of some 40 residues. The variable regions coin­ cided with the maxima of hydrophilicity. One of the variable Figure 1 shows the polypeptide chain fold as a ribbon diagram regions was known to be surface exposed [54, 55]. Using mono­ of porin from Rhodobacter capsulatus, porin from Rhodopseudo­ clonal antibodies against the other variable stretches, Tommassen monas blastica and OmpF and Phoe from Escherichia coli, respec­ could show that all except the third form surface exposed tively. The common feature is a transmembrane /3-barrel of epitopes. Infrared studies had previously demonstrated that por­ approximately 30 A in diameter. It is formed by 16 antiparallel ins contain a high degree of antiparallel /3-pleated structure with /3-strands which are connected to their next neighbors. The N- and strands of average length of 11 residues oriented roughly parallel C-termini are located near the periplasmic side [54, 59, 84]. The to the membrane normal [56, 57]. With these data, Tommassen strands are inclined to the barrel axis on average by 4SO [76]. The proposed that the polypeptide forms /3-strands crossing the mem­ connections between the strands are of the short hair-pin type on brane 16 times in an antiparallel manner which are connected on the periplasmic side and longer and more irregular on the external the surface by the eight variable loop regions. On the peri plasmic side. The latter account for the hydrophilicity peaks and variable side are the N- and C-termini and seven hairpin loops connecting sequence zones which Tommassen had observed. The three porin neighboring strands. The same analysis was applied to class 1 to 3 structures show that this sequence variability goes parallel with a meningococcal and to PIA and PIB gonococcal porins, and structural variability in these loops and in the contour of the upper indicated a similar structure for these porins as well [58]. This barrel rim. One extreme is represented by the porin from model coincides remarkably well with the structures found lately Rhodobacter capsulatus 37b4. Near the trimer axis and the termini by X-ray crystallography. It also gives hints how the sequence can the barrel wall height is about 20 A. At the opposite circumfer­ be used to predict the folding of porins of unknown structure in ence, which forms the periphery of the trimer, it is ca. 50 A. The absence of homology with porins of known structure [59]. For external end of the 0 trimer, as a consequence, forms a single VDAC, models consisting of a N-terminal helix and 19, 16 or 12 channel of about 50 A internal diameter which ends at its bottom anti parallel transmembrane /3-strands have been proposed [60- in three narrow constriction sites (eyelets) near the bilayer center. 62]. Beyond each eyelet one widening channel formed by one porin molecule runs further to the periplasmic surface. In the other Structural studies porin structures this fusion of three molecular channels to one Matrix (OmpF) porin was the first integral membrane protein trimer channel is much less obvious, so a monomeric porin of which single crystals suitable for X-ray diffraction could be channel would also appear to be conceivable. 932

A B

Fig. 1. Ribbon diagrams showing the polypeptide chain fold and secondary structure of (A) porin from Rhodobactcr capsulatus 37b4, (B) porin from Rhodopseudomonas blastica and (e) OmpF and PhoE from Escherichia coli. The view is from the trimer axis towards the eyelet. This and the following figures were prepared with the program Molscript [121] on Silicon Graphics workstations. 933

Fig. 2. Ribbon diagram of the trimer of porin from Rhodobacter capsulatus 37b4. Several 2 hydrophobic amino acids and the bound Ca + atom in the molecular interface (black ball) as well as two bound Ca2 + atoms at aspartate and glutamate residues of loop L3 are shown. The view is from the external medium.

In OmpF, some of the external loops tend to incline towards the is in accord with what was said above about porin export and trimer axis to constrict the external channel entrance. The differ­ membrane insertion. ences in the external loop structure between Enterobacteria and Purple bacteria could have evolved due to the different environ­ The membrane embedded suiface mental risks to which these cells are exposed in their habitats. Figure 3 shows a ribbon diagram of the porin from Rhodobacter In contrast to the other external loops, the third loop L3 of ca capsulatus 37b4, as seen from the external surface. All porin 45 residues folds in all porins into the barrel along the wall structures show two rings of aromatic acids around the trimer approximately in the center of the bilayer and then back to the peripheral surface, that is, outside the intermolecular contact sixth strand. Its interactions with peripheral barrel strands may surfaces. One ring is close to the smooth periplasmic bottom rim. contribute to the stability and form of the barrel [81]. Phenylalanines and occasionally tryptophanes with their aromatic rings always pointing towards the hydrophobic phase and ty­ The molecular inteiface rosines with their hydroxyl groups always pointing towards the Figure 2 shows the trimer center of porin from Rhodobacter aqueous phase are found on the first strand position which follows capsulatus. In the trimers of all porins, the N- and C-termini are or precedes one of the hair-pin loops. At a distance of ca. 15 A packed tightly together into the hydrophobic intermolecular con­ towards the external surface, a second, parallel ring is formed by tact interface near the trimer axis. Its tight packing is indicated by tyrosines and tryptophanes. In between, only nonpolar residues low temperature factors around 20 A2 [78, 82]. Its energy of like Leu, Val, Ala and lIe are found. The resulting hydrophobic formation may stabilize trimerization and membrane incorpora­ zone has an axial height of ca 25 A, approximately sufficient to tion [78]. This could be triggered by the signal sequence cleavage span the 30 A thick hydrocarbon layer of lipid bilayers [85, 86]. As [50]. In case of the porins from Rhodobacter capsulatus and the thickness of the hydrophobic layer may change due to Rhodopseudomonas blastica, N- and C-termini of neighboring gel-to-liquid phase transition, which depends upon parameters as porin molecules contribute to the interaction by forming salt temperature, pH, divalent ion concentration and temperature [87] bridges. In the former porin a Ca2 + -binding site, formed by and as the protein may rock upon thermal movements, the adjacent molecules, also contributes to trimerization (Fig. 2) position of the hydrophobic-hydrophilic interfaces on the protein [77-79]. In the two Escherichia coli porins one of the external surface may change. Porins thus may have to accommodate to loops near the trimer axis folds to the barrel rim of a neighboring such fluctuations. The aromatic rings may confer this adaptability. molecule and seems to connect both. These features of the Two mechanisms have been proposed: the polarizability of the interface explain the extraordinary stability of porin trimers which 7T-electron system allowing interaction with polar- and apolar can be dissociated only upon boiling in sodium dodecylsulfate and solvents [88, 89] and rotations of the aromatic side chains relative 934

Fig. 3. Ribbon diagram of porin from Rhodobacter capsulatus 37b4 with aromatic amino acid residues. The membrane-embedded surface with the two rings of aromatic residues is shown.

to the backbone [82]. These well-conserved aromatic amino acid ions cannot pass it freely. Figure 5 shows the constriction site of residues can be used for structure predictions of porins [59] and the Rhodobacter capsulatus 37b4 porin with its charged amino­ are found less clear also in reaction centers [90, 91] and bacterio­ acid residues superimposed with the diameter of a hydrated K-t rhodopsin complexes [92]. Beyond the external aromatic ring the ion. The diameter was calculated using the known number of peripheral barrel surface bears some carboxyl groups, which could bound water molecules and the volume of a water molecule [93]. interact with lipopolysaccharide molecules through divalent cat­ As porins allow fast movements of ions across the membrane with ions [77, 81]. the pore conductance beeing proportional to the bulk aqueous conductivity for many electrolytes [15], there must be a compen­ The constriction zone sation of the energy penalty of transiently stripping some of the In all known porin structures, the space filled by the loop L3 hydration water. Various evidences exist that interaction of cat­ along its path inside the barrel wall effectively reduces the free ions with acidic and anions with basic residues provide this energy. cross section in the center of the membrane to a constriction site The ion selectivity is pH dependent [94, 95]. Furthermore, porins or eyelet of, typically, 8 A by 10 A. Figure 4A shows a 4 A slab with acetylated or carbamylated lysines show increased cation from Rhodobacter capsulatus 37b4 porin close to a plane spanned selectivity [95, 96]. If hybrid porins of a cation- and a anion­ by the trimer (membrane normal) and a line in the membrane selective one are made, anion selectivity changes into cation plane from the trimer axis to the center of the constriction site. selectivity upon exchange of a lysine by an aspartate [97]. Ex­ The constriction of the barrel by the loop L3 is obvious. The change of severallysines for glutamates in PhoE each resulted in surface in this channel segment is studded with charged amino a cation selective channel [98]. When the constriction sites of acids and tyrosines. In porin from Rhodobacter capsulatus 37b4, OmpF (moderately cation selective) and PhoE (moderately anion there are 7 Asp and 4 Glu contributed by L3 and 2 Lys, 3 Arg and selective) are compared, the latter is found to possess two 1 His eontributed by the opposite barrel wall which is adjacent to additional lysine residues in the central constriction zone [81]. the trimer center. In the two Escherichia coli porins the external The exact mechanism of ion selectivity, the role which single loops L4 and L5 are inclined towards the barrel axis, so that their charged residues play, will probably require further theoretical charged amino acids form a further constriction near the channel analyses of the known structures. entrance as seen in Figure 4C. The constriction zone also determines the size exclusion limit as L3 constricts the barrel cross section to an extent that hydrated was concluded [81] from mutants of Ompe with an increased 935

A B

•

c

Fig. 4. CPK slabs of between 0.6 and 5 A thickness cut along a plane through the trimer axis and the center of the eyelet for (A) porin from Rhodobacter capsulatus 37b4, (B) porin from Rhodopseudomonas blastica and (C) OmpF and PhoE from Escherichia coli. Backbone atoms are drawn as open circles (0), side-chain atoms as filled circles (e). As a visual aid, the shape of the barrel is indicated by a cylinder in A. The cross section together with the barrel walls is seen, the low-height wall near the trimer axis is on the left side. The constriction is caused by the two branches of loop L3 which are attached to the inner side of the peripheral wall. From the constriction towards the external surfaces, the channel is widening, although differently in the four porins: the channels of OmpF and PhoE show a further constriction near the external entrance of the channel.

exclusion limit which were obtained after applying a strong their mutual repulsion. This indicates that they are held in selection pressure by growth on large molecular weight maltodex­ position by a strong electric field. The water ordered by this field trins [99] and from site-specific mutations of PhoE [100]. The must be structured and thus cause a high energy barrier for constriction zone seems to function also as a filter against apolar passage of small apolar substances [82]. This reminds of a similar substances. The arginine and lysine residues are aligned parallel in barrier function of the long oligosaccharide chains of lipopolysac­ van der Waals contact with low temperature factors in spite of charides [4]. 936

constriction and that other loops of the external barrel rim constrict the external channel entrance and alter their conforma­ tion upon gating. In VDAC, several amino acids distributed over the whole polypeptide were found to be responsible for voltage gating [103]. Together with the large exclusion limit this also indicates a different folding of the loops as compared to the known bacterial porin structures. The eyelet construction as well as the gating mechanism in these porins thus should not be assumed to be universal to all porins.

Bound substances Detergent binding has been suggested from cylindrical electron density in the inside barrel wall of porin from Rhodobacter capsulatus [78] as well as near the trimer axis at the external end of the barrel of Rhosopseudoman blastica [S2]. Two Ca2 + -binding sites have been found in the region of loop L3 rich in acidic amino acids [78]. Fig. 5. Ribbon diagram of porin from Rhodobacter capsulatus 37b4, seen from the external side. In addition, the charged residues of the narrow part Possible relevance of the porin structure for other membrane of the channel are shown: 5 basic residues and 1 His attached to the wall proteins side near th~ trimer axis. and 9 acidic residues, mostly attached to lool? L3. For companson, the dIameter of a hydrated potassium ion (14 A) is Where else could (3-barrels in proteins [104] be used as surfaces indicated by a circle. of channels?

Channels with barrels wider than those of porins

As the arrangement of charged residues around the constriction A class of outer membrane proteins is formed by the receptors site seems to be critical for high rates of ion permeation through for siderophores (iron chelating complexes), FhuA, FepA and the porin channel, the voltage-induced partial closing of porins FecA and the vitamin B12 receptor [105]. Recently, for FhuA a could be due to a field-induced conformational transition disturb­ 32-stranded l3-barrel has been proposed [106]. One loop forms a ing the structural arrangement of charges of these residues. In binding site for ferrichrome and its deletion renders the protein a Figure 6 the positions of these residues are shown when viewing large open channel [107]. Similar results were obtained with FepA from the trimer axis towards a porin monomer from Rhodobacter for which a 29-stranded barrel was proposed [lOS]. capsulatus 37b4. The center-of-mass of negatively charged resi­ A large transmembrane l3-barrel was also proposed for the dues is slightly shifted towards the external surface as compared to membrane-inserted oligomer of a bacterial toxin, aerolysin, [109]. the center-of-mass of the positively charged residues. In voltage­ Channels with barrels narrower than those of porins gating experiments the electric field is applied parallel to the membrane normal and is thus either pushing the opposite charges OmpA, an outer membrane protein from Escherichia coli with towards each other or pulling them apart. In the former case, the 325 residues, consists of a 177 amino acids long N-terminal external field adds to the mutual attraction of the charges. domain for which an S-stranded (3-barrel has been predicted Consequently, a breakdown of the local arrangement is feasible which seems to be linked flexibly to a C-terminal periplasmic with a concomitant shift of opposite charges towards each other. domain [110, 111]. OmpA has been shown to form pores of low The shift could be brought about by a swing of the extended side permeability but similar exclusion limit as general diffusion porins chains of Asp, Glu, Arg and Lys towards each other, the former [112]. swinging slightly towards the periplasmic-, the latter towards the Barrels in water-soluble binding-proteins of known structure external surface. It could also be the case, that L3 is to a certain consist of S or 10 strands. The retinol-binding protein [113] is a S degree detached by the applied force from the barrel wall and its stranded barrel, 10 stranded barrels are found in the cellular mobile part swings towards the opposite barrel wall. retinol binding protein and the P2 myelin protein [114]. By their This view is in accord with the experiments of Brunen and narrow inner diameter the latter are suited to form well-adapted Engelhardt [28] who observed an increase of gating voltage upon geometrical binding sites for small molecules. These narrow titration to high or low pH values. The neutralization of charged barrels could be used as channel domains of specific ion channels. amino acid residues would decrease the attraction of the oppo­ In addition, a high symmetry of the barrel could be advantageous sitely charged parts, so that a higher external field is needed to to achieve specificity [115]. Eight-stranded symmetrical barrels cause the breakdown of the arrangement. have indeed been proposed for a dimeric Cl- channel [116] and However, in porin from Haemophilus influenzae a residue in the for tetrameric voltage gated ion channels [117, I1S]. fourth external loop (Arg 166) was found to be critical for gating [27]. This porin has a significantly larger exclusion limit as Channel geometry and channel properties compared to the four porins of known structure of 1400 Da [101], The general diffusion porins with their 16-stranded barrels have reminiscent to protein F of Pseudomonas aeruginosa [102]. This both a wide entrance and outlet with a constriction site halfway in may indicate that loop L3 in this porin does not form an eyelet between. With this geometry they minimize frictional interaction 937

Fig, 6. Simplified ribbon drawing of porin from Rhodobacter capsulatus 37b4 showing the charges of amino acids near the constriction site as seen from the trimer axis. The center of mass of the negative charges is slightly shifted to the external side as compared to the positive charges.

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