Structures of membrane proteins

Abstract

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class.

Fig. 1

Progress of membrane protein structure determination. Starting with the first structure in 1985, 174 unique membrane protein structures have been determined till the end of 2009. However, the Protein Data Bank (PDB) holds many more than this with for example, over 60 coordinates each for reaction centres and bacteriorhodopsin alone. We have included in the chart only polytopic membrane proteins that have a functional role within the membrane and not intrinsic membrane proteins with only a single, presumably regular trans-membrane α-helix. Mutants, different conformational states, structures with bound substrates/inhibitors of the same protein, or membrane proteins from different species with >70% sequence homology are counted only once. There are numerous ways of classifying membrane protein structures: here we present the distribution classified on the basis of α-helical or β-barrel secondary structure; a different classification on the basis of prokaryotic or eukaryotic origin can be found elsewhere (Carpenter et al. 2008). In the early years, structures were determined from proteins that were abundant in their natural environment including the reaction centres (1985 and 1987), bacteriorhodopsin (1990), porins (1992), light harvesting complex (1994) followed by a variety of electron transport and photosynthesis complexes. The first structures of membrane proteins expressed recombinantly started to emerge from 1998 (KcsA, MscL, OmpA and FhuA). Since then, the availability of sequenced genomes in the late 1990s propelled the rate of membrane protein structure determination, which has reached its highest level in the past two years. The following link provides a complete list of available structures with links to the PDB (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). In this review, we give the PDB accession number for the structures shown in the figures.

Fig. 2

Tetrameric potassium channels: (a) KcsA (1K4C) and (b) Kv1.2/Kv2.1 chimera (2R9R) with extracellular side at the top. Potassium ions are shown as red spheres. The prokaryotic channel KcsA represents the simplest potassium channel with two TM helices. Each subunit is coloured individually. The extracellular (top) section of the pore has a stretch of β-structure conserved by evolution for potassium ion selectivity. The gating of KcsA is thought to occur by a change in pH, with the channel opening at acidic pH (Heginbotham et al. 1999). Many other K⁺ channels have a more complex architecture with six TM helices. The structure of the Kv1.2/Kv2.1chimera (Long et al. 2007) is one such example. The pore forming helix S5 and outer helix S6 are coloured as in KcsA. The voltage sensing S4 helix (dark blue) is replete with arginines that are thought to move in response to a change in membrane potential resulting in opening or closing of the channel. Voltage-gated K⁺ channels also have a β-subunit (light orange) that is essential for regulation and makes contact with the TM domain via linker T1 (brown). The structure of KcsA depicts a closed channel while the Kv1.2/Kv2.1 chimera is probably an open depolarised state.

Fig. 3

Pentameric ligand-gated channels, side view of full-length protein and top view of TM domain only: (a) AchR (2BG9), (b) ELIC (2VL0) and (c) GLIC (3EAM). Ligand-gated channels are non-selective cation channels that form homo or hetero pentamers. Each subunit in the figure is coloured individually. In contrast the major voltage-gated channels are tetrameric (shown in Fig. 2) and are selectively permeable to K⁺ or Na⁺ ions. In higher organisms, ligand-gated channels play a major role in signalling, the best-known example being the acetylcholine receptor (AChR) in the neuromuscular junction. The recent identification of prokaryotic pentameric ligand-gated channels (ELIC and GLIC) through genomic homology searches has allowed the determination of two high-resolution X-ray structures but their physiological function remains to be elucidated. Each subunit of all channels in this family has four TM helices. A large extracellular domain binds the ligand, acetylcholine in AChR, protons in GLIC and unknown in ELIC. In AChR, the pentamer composition is α₂βγδ-subunits with only the two α-subunits binding the ligand. Structures of AChR determined by EM and ELIC by X-ray crystallography reflect the closed state of channel, while GLIC crystallised at low pH is probably an open state of the channel. The outward tilting of the inner helices in GLIC, proposed as the basis of channel opening, can be seen in the top view.

Fig. 4

Gap junction (2ZW3): (a) side view of complete gap junction and (b) top view (from cytoplasm) of a hemichannel. Gap junctions are made up of connexin monomers that assemble into hexameric rings called connexons. A complete structure of a gap junction consists of two apposed connexons mediated by strong interactions between the extracellular domains and a continuous open channel that connects the cytoplasm of adjacent cells. A connexin monomer [highlighted in colour in the side view (a)] has four TM helices with surface loops connecting them. TM1 (dark blue) and TM2 (light blue) form the wall of the pore. TM3 and TM4 (light orange) form the outer helices. The extracellular loops 1 and 2 (aquamarine) form most of the interactions between the two interacting connexons and seal the junction from the extracellular environment. The putative voltage sensing N-terminal helix is shown in salmon.

Fig. 5

Trimeric ion channels: (a) ASIC (3HGC), (b) P2X receptor (3H9V). Trimeric ion channels have thus far been found only in eukaryotes. Acid sensing ion channels (ASICs) and P2X receptors belong to the family of voltage-independent, ligand-gated cation channels. In contrast to the pentameric ligand-gated ion channels, ASIC and P2X receptor are trimeric. They have the commonly found architecture of two TM helices (blue, yellow and green) connected to a large extracellular domain (light green) that binds ligand. One salient feature in the extracellular domain of both these channels is the presence of a large number of cysteines that form disulphide bonds (red sticks), which is postulated to provide rigidity during conformational change upon ligand binding. The ligand in the case of ASCI is a proton, and for P2X it is ATP. The structures of ASIC and P2X receptor represent the closed states of the trimeric ion channels, since they have been crystallised at low pH and in the absence of ATP, respectively.

Fig. 6

Molecular architecture of prokaryotic mechanosensitive channels, side and top view: (a) MscL, closed (2OAR), (b) MscS, closed (2OAU), (c) MscS, open (2VV5). MscL and MscS are non-selective channels, activated in response to hypo-osmotic shock. MscL and MscS show unusually large conductances of 3 and 1 nS, respectively, which are much larger conductances than found in ion-selective channels because they make very large transient holes in the membrane. Although they carry out similar functions, the structures of these proteins are remarkably different indicating separate evolutionary pathways. MscL is a pentamer with two TM helices in each subunit, while MscS is a heptamer with three TM helices per monomer. One subunit in each channel is coloured in rainbow, blue at the N-terminus and red at the C-terminus. Despite their differences in oligomeric state, both proteins show a ring of single TM helices tightly packed to form the permeation pathway and covered by loosely packed outer helices. There is little sequence conservation between the pore forming helices of these two families of proteins. However, there is a striking common feature in the central helices of the two channels. When TM1 of MscL and TM3 of MscS are compared, there is a conserved pattern of alanine and glycine residues that allow tight packing of the pore forming helices, with interspersed hydrophobic residues that form the constriction pathway when the channels are closed. The presence of small amino acids in these helices must play a pivotal role in facilitating structural changes during gating. Indeed, the open structure of MscS shows a large rotation and tilting of helices, which results in the increase of pore diameter from 4·8 Å in the closed state to ~13 Å in the open state. Cross-linking and site-directed spin labelling studies indicate that MscL probably undergoes a similar conformational change to open the permeation pathway, creating a pore diameter of ~25 Å.

Fig. 7

(a) Tetramer of aquaporin (2B60). (top) Aqp0, determined by electron crystallography viewed from the extracellular side, shows an individual water channel in each monomer with water molecules depicted as red spheres. Lipids probably fill the cavity at the centre of the tetramer. So far only in the structure of human Aqp5, has an ordered lipid molecule been identified in this region (Horsefield et al. 2008). (b) Monomer of aquaporin showing how it has evolved by gene duplication. The N and C terminal halves (blue and green) of each polypeptide are related by a pseudo twofold symmetry axis, which is parallel to the membrane plane and perpendicular to the page. Thus, the two halves of the molecules have opposite orientations in the membrane. Loop C (magenta) on the extracellular side connects the two halves. The conserved NPA motifs and short helices (HB and HE) are depicted in yellow. Three water molecules (red spheres) as seen in the EM structure of Aqp0 are shown along the pore region (Gonen et al. 2004b). The hourglass like structure of aquaporin was predicted based on primary sequence and biochemical analysis years before the first structure was determined (Jung et al. 1994).

Fig. 8

Ammonia channel (1U7G). (a) Trimeric ammonia channel, viewed from the extracellular side, showing an ammonium ion (red) at the periplasmic entrance and three ammonia molecules (yellow) in the lumen. One monomer coloured as in (b) clearly shows the pseudo twofold symmetry. The trimer of the ammonia channel is very stable even in the presence of SDS and tight packing between monomers is possibly the reason why the crystals are so well ordered. Among the membrane protein structures known so far, this eleven helical bundle is unique to the ammonia channel family. (b) Monomer of the ammonia channel: Unlike the aquaporins, the sequence of AmtB does not reveal any hint of gene duplication, but the structure shows that helices 1–5 (blue) and 6–10 (green) are related by pseudo twofold symmetry. Like aquaporins the pseudo twofold axis lies in the membrane plane so that the two halves of the molecule have opposite orientations in the membrane. Together they make the pore. Helix 11 (magenta) lies at a 45° angle to the membrane and makes contact with both halves of the monomer.

Fig. 9

Molecular architecture of P-type ATPases shown with the cytoplasmic active site at the bottom, using the convention adopted throughout this review. (Note that in the P-type ATPase field, the orientation is normally inverted from that shown here). (a) Ca²⁺-ATPase (2ZBD) and (b) Na⁺/K⁺-ATPase (2ZXE). The formation of an acid-stable phosphorylated intermediate during the transport cycle of this class of membrane proteins gives rise to their name, P-type ATPase. All proteins in this family have three cytoplasmic domains, a nucleotide binding domain (green), a phosphorylation domain (yellow) and an actuator domain (blue). The TM domain (brown) with 10 α-helices is the least conserved of all domains. The β-subunit of Na⁺/K⁺-ATPase is shown in pink and the γ-subunit (FXYD protein) in orange. The transport cycle of P-type ATPases has been described by Post and Albers (Albers et al. ; Post et al. 1965) to alternate between E1 and E2 states with different affinities for ions and nucleotides. Two representative structures of Ca²⁺-ATPase and Na⁺/K⁺-ATPase show a general architecture preserved in P-type ATPases. It has been possible to trap intermediates in the transport cycle by the use of transition state analogues and inhibitors with ATPase. The E1P state of Ca²⁺ ATPase (a) with calcium ions (cyan) occluded within the protein was obtained by the addition of AlF₄⁻ (aquamarine) and Mg.ADP (orange). In the Na⁺/K⁺-ATPase (b), when a phosphate mimic such as MgF₄²⁻ (magenta) is crystallised with counterion potassium (purple), it was possible to trap the pump in the ion-occluded E2 state. For a complete discussion of multiple structures and conformational changes, see a recent review (Toyoshima, 2008).

Fig. 10

Light driven pumps: (a) bR (1FBB and 1FBK) and (b) sRII:HtrII dimeric complex (1H2S). Bacteriorhodopsin is one of the simplest monomeric membrane proteins, composed of a seven-TM helix bundle with the chromophore retinal (red) bound in the centre of the bundle. Light isomerises the retinal and a proton is then released towards the extracellular side from its Schiff base with K216 (blue). A large structural change, (from purple to yellow in panel a) then opens the cytoplasmic half-channel to allow reprotonation of the Schiff base and completion of the cycle. A similar structural change is thought to be present in other members of the bacteriorhodopsin family. Here the structure of sRII, a photophobic sensor (orange) in halobacteria is shown in complex with the helical hairpin dimer (green) of its cognate transduction partner, HtrII. In this case, the structural change couples to a cytoplasmic chemotactic/phototactic methylation cascade.

Fig. 11

Side and top view of the rotor subunits from prokaryotic V-type or F-type ATPases: (a) K-ring (2BL2) and (b) c-ring (1YCE). The rotors of the F₁F_o and V₁V_o-ATPases comprise multimers of identical subunits that give rise to c-rings and K-rings, respectively. The c-ring of Ilyobacter tartaricus is an 11-membered ring in which each monomer is a helical hairpin (two subunits are highlighted in cyan) with a conserved glutamate or aspartate residue (red sticks) in the middle of membrane. The K-ring of Enterobacter hirae is a 10-membered ring, with each monomer having two homologous hairpins (cyan and yellow) covalently linked resulting in four TM helices per protein monomer. Only one of these hairpins has the conserved glutamate (red sticks in subunit coloured yellow) resulting in a lower ion to ATP ratio than would be obtained if both hairpins retained a glutamate. However, the structure is equivalent to a 20-membered c-ring. Both proteins are sodium-dependent ATPases and the crystal structure shows bound sodium ions (purple) in all subunits. Residues from the same subunit mediate sodium coordination in K-ring, whereas in the c-ring residues from neighbouring subunit contribute to sodium coordination. The top view of both proteins shows a central hole filled by lipids (green sticks).

Fig. 12

ABC (ATP-binding cassette) exporters (a) P-glycoprotein (3G61) and (b) Sav1866 (2HYD). Based on directionality, ABC transporters can be classified as importers or exporters, which use the energy of ATP to import or export substrates. In ABC exporters, the TM (blue and yellow) and nucleotide-binding domains (light blue and light yellow) are expressed either as a single polypeptide, which then associates to form a dimer as in Sav1866 (b) or as one single large polypeptide as in P-glycoprotein (a). The two structures of ABC exporters show an inward facing (a) and outward facing conformation (b). P-glycoprotein crystallised in the presence of substrates, but in the absence of nucleotide, adopts an inward facing conformation with the nucleotide-binding domains spread apart. Substrate (green) binds at an interface between the two halves of the TM domain. In contrast, Sav1866 crystallised in the presence of ADP (orange), reveals an outward facing conformation with the nucleotide-binding domains in close contact. The substrate for prokaryotic Sav1866 is unknown.

Fig. 13

ABC importers (a) MetNI (3DHW), (b) ModABC (2ONK), (c) MalFGK (2R6G), (d) BtuCD (1L7V) and (e) HIF (2NQ2). Found only in prokaryotes, the domains of importers are encoded and assembled separately (TM domains are in blue and yellow, and the nucleotide-binding domains are in red). Additional regulatory domains can be also found within the NBD such as the C2 domain (cyan) in the MetNI transporter. A high-affinity periplasmic binding protein (green) delivers the substrate to the transporter (ModABC and MalFGK). Based on the TM architecture ABC importers can be subdivided into type I and II importers. Type I importers have a variable number of TM helices (10–14) but the core helices as defined by the minimal MetNI methionine transporter is conserved (a). More commonly observed are 12 TM helices (ModABC and b) while maltose transporter MalFGK has additional peripheral helices (c with additional helices shown in grey). The structure of MetNI (a) without a substrate or nucleotide, and the structure of the molybdate/tungstate transporter (b) from A. fulgidus, with binding protein and substrate (tungstate in magenta) but in the absence of ATP, both reveal inward facing conformations. In MalFGK (c), the presence of ATP (orange) and the binding protein induces an outward facing conformation of the TM domain while the substrate (maltose in magenta) having been released from the binding protein is occluded (Oldham et al. 2007). Type II importers transport larger substrates such as vitamin B₁₂ and chelated metal. Examples include BtuCD, the vitamin B₁₂ transporter and the homologous HI1470/71, which transports chelated metal. Each monomer has 10 TM helices. BtuCD and HI1470/71 were crystallised in the absence of substrate or nucleotide but reveal outward and inward facing conformations, respectively, probably induced by the detergent environment (Locher et al. ; Pinkett et al. ; d and e). With all these structures, one can envision a productive cycle, which is described in the main text. However, in the absence of structures from the same protein and considering the probable effects of detergent (BtuCD and HI1470/71), more biochemical and structural data is required to obtain a complete picture of the transport cycle.

Fig. 14

Structural gallery of some secondary transporters (a) ATP/ADP carrier (1OKC), (b) LacY (1PV7), (c) EmrE (3B5D) and (d) ClC (1KPL). Secondary transporters make up one of the largest membrane protein families, which is dynamic and diverse. Like many membrane proteins, secondary transporters evolved by gene duplication and fusion. Such repeats are clearly revealed in the recent structures of secondary transporters. The interface between the repeats forms the substrate binding site and translocation pathway. In the ADP/ATP translocase (a), a mitochondrial carrier, the simple repeat is made up of ~100 amino acid helical hairpins that have been triplicated (blue, green and yellow) to give a six-TM helical protein with a pseudo threefold axis relating the hairpins. This structure of ADP/ATP translocase with bound inhibitor carboxyatractyloside (red) shows an outward facing conformation. Duplications of helical bundles are more generally observed resulting in either parallel or antiparallel orientations in the membrane. Lactose permease, LacY (b), a major facilitator superfamily (MFS) transporter has evolved by gene duplication of 2 six helical bundles (blue and yellow) with the same orientation in the membrane. This architecture gives rise to a cavity where substrate (red) binds. Many transporters have their duplicated domains assembled in an antiparallel orientation resulting in two halves wrapped around a common centre bringing amino acids from distinct α-helices together. Occasionally, such duplicated domains are expressed as individual polypeptides and assembled to form an oligomer. EmrE, a small multi-drug transporter (c), is synthesised as two polypeptides (blue and yellow) which are inserted into the membrane in antiparallel orientation, to make an asymmetric homodimer, with inhibitor/substrate tetraphenylphosphonium (red) binding at the monomer-monomer interface. The chloride/proton antiporter, ClC (d), is one such transporter in which the duplicated domains (blue and yellow) are antiparallel in orientation with chloride ions (green spheres) binding at the interface.

Fig. 15

Two sodium-dependent transporters (only monomers are shown) (a) GltPh (2NWX) and (b) NhaA (1ZCD). Sodium-dependent transporters have characteristic breaks in their TM helices and these breaks are crucial for ion binding. The architecture of sodium-dependent transporters differs significantly (see text and compare with Fig. 14). The glutamate transporter (GltPh) from Pyrococcus horikoshii and the sodium/proton antiporter (NhaA) from E. coli are two examples of sodium-dependent transporters. GltPh is a homologue of the mammalian excitatory amino acid transporter family. It forms a trimer but each monomer has a separate transport pathway. The monomer of GltPh comprises eight TM helices and two, opposite facing, helical re-entrant hairpins (blue and yellow). Of the eight TM helices, only two (green) associate with the hairpins to form the transport pathway while remaining six helices (grey) form contacts with other monomers. Two sodium ions (violet) and substrate aspartate (red) are found in the transport pathway with residues contributed from the two hairpins and TM helices 7 and 8. Conformational changes in the helical hairpins, TM7 and TM8 are thought to accompany substrate transport. In contrast, NhaA comprises 12 TM helices with two distinctive domains. Six TM helices are involved in transport (blue and yellow) with two discontinuous helices exposing their main chain carbonyls oxygens being responsible for ion co-ordination. The remaining helices (grey) provide a supporting structural role. Unlike in some other transporters (such as those shown in Fig. 14), gene duplications in both GltPh and NhaA are confined only to helices involved in transport and not the entire protein.

Fig. 16

Different conformational states of sodium dependent secondary transporters: (a) Mhp1, hydantoin transporter (2JLN), (b) LeuT, leucine transporter (2A65), (c) BetP, betaine transporter (2W8a) and (d) vGlt1, galactose transporter (3DH4). Different conformations have been observed in structures of the sodium-dependent transporters Mhp1, LeuT, BetP and vGlt1. Although, these transporters show little sequence homology a core structure of 10 TM helices (blue and yellow) involved in substrate translocation share a recognisably similar fold. Additional helices (grey) might have evolved for specific needs or modulation. The structure of Mhp1 with no substrate reveals an outward facing conformation, while LeuT with leucine (red) and two sodium ions (purple) reveals a substrate-occluded but outward facing state. BetP with substrate bound (red) probably represents an intermediate occluded state with both gates closed. In contrast, vGlt1in the presence of galactose (red) and a sodium ion (purple) reveals an inward facing conformation. These structures indicate the existence of multiple states, but it is not clear whether a particular transporter has its own preferred state to crystallise or the presence of detergent, substrate or crystallisation conditions modulates the series of states in the transport cycle. Discontinuous helices in all four transporters are involved in substrate or sodium ion binding. Conformational changes are thought to occur in the discontinuous helices accompanied by changes in extracellular and intracellular gates resulting in substrate translocation.

Fig. 17

Protein translocation channels: (a) SecYEβ (1RHZ) and (b) SecA–SecYEG (3DIN). Monomeric SecYEβ in M. jannaschii forms the minimal protein translocation unit. The channel forming SecY is made up of 10 TM helices that can be divided into two equal halves (blue and yellow) related by a pseudo twofold symmetry axis parallel to the plane of the membrane. Like many other membrane proteins, the two halves thus have an antiparallel orientation. SecE has a short amphipathic helix and a very long highly tilted TM helix (green) and forms extensive contact with SecY. The β-subunit (purple) is oriented perpendicular to the membrane and lies just outside SecY. Together SecE and Secβ are thought to provide structural integrity to SecY during protein translocation. A small helix (red) acts as a plug to seal the channel when no protein is translocated. A complex between SecA and SecYEG of T. maritima was obtained in the presence of ADP and BeCl₂. SecG of T. maritima (equivalent to the β-subunit of M. jannaschii) has an additional TM helix (purple). The five domains of SecA are coloured individually. The two nucleotide-binding domains are coloured wheat and grey with a molecule of ADP shown in black. The polypeptide-crosslinking and helix wing domains are shown in aquamarine and orange, respectively. The helical scaffolding domain (brown) of SecA makes contact with the C-terminal domain of SecY. A comparison of the two structures reveals movement of three TM helices in SecY, which opens up the central channel and is accompanied by displacement of the plug helix (red). Channel opening allows the polypeptide to be secreted either to the periplasm or laterally into the membrane.

Fig. 18

Electron transport chain: (a) complex II (1ZOY), (b) complex III (2FYU) and (c and d) complex IV (1V54). (a) Complex II, functional as a monomer as illustrated, shows four subunits of which two are membrane spanning each with three TM helices (blue and yellow), together with five cofactors, of which only the haem b (red) and the quinone binding site (not shown) are located at the level of the membrane. FAD (orange) and three iron–sulphur clusters (brown) are also shown. (b) Complex III, showing only one half of the dimer that has the TM helix of the ISP-Rieske subunit (cyan) domain swapped with its TM helix (protruding on the left side of the molecule) interacting more closely with the other monomer. There are eight subunits in the bovine mitochondrial enzyme, of which the cytochrome b subunit is most important (blue). It contains eight TM helices in two bundles, of five and three helices. The five-helix bundle forms the binding site for the two b-type haems (red) that are aligned vertically above one another with each haem iron being coordinated by two histidine side chains that are 14 residues apart (four turns) on helices B and D. The other three helices of cytochrome b and the remaining five single TM helices from other subunits have peripheral roles (grey). Cytochrome c₁ (yellow) with its c-type haem (brown) is located in the intramembrane space. The cytoplasmic/matrix subunits (grey) are also not directly involved in electron transport. (c) Complex IV, showing a side view of only the two functionally important subunits I and II (blue and yellow), together with the three metal centres: the Cu_A two-copper site in subunit II (brown spheres), and the sites for haem a (red) and binuclear haem a₃/Cu_B (red and green) in subunit I. (d) Top view of complete monomer of bovine mitochondrial complex IV, showing all 28 TM helices in cross-section. Only the 12 helices (blue) of subunit I and the two helices of subunit II (yellow) are directly involved in electron transport and proton pumping. These 12 TM helices in subunit I are related by an approximate threefold axis, easily visible in the picture with haem a (red) being located in one sector, haem a₃/Cu_B (red and green) being located in the second sector and proton channel D being located in the third.

Fig. 19

Photosynthetic complexes: (a) reaction centre (1PRC), (b) RC-LH1 complex (1PYH), (c) PSI complex (2O01) and (d) PSII complex (3BZ1). The common reaction centre (RC) core can be seen here in the same orientation in each part of the figure. In (b), (c) and (d), the RCs are surrounded by a great variety of subunits (see text). The oxygen-evolving centre (OEC) is shown as pink spheres in PSII. Choloropyhll in all proteins are shown as grey sticks.

Fig. 20

G-protein-coupled receptors: (a) bovine rhodopsin (1GZM), (b) β₁-adrenergic receptor (2VT4), (c) β₂-adrenergic receptor (2RH1) and (d) adenosine A_2a receptor (3EML). These four structures all represent inactive, antagonist-bound, conformations of four different G-protein-coupled receptors. The structures are related as they all have a homologous seven-helix TM bundle, but differ in the details of their binding sites and structures at both cytoplasmic and extracellular surfaces. A hypothetical mechanism of activation and signalling to the cytoplasmic αβγ G-protein complex is described in the text.

Fig. 21

Structure of intramembrane proteases: (a) rhomboid GlpG (2IC8) and (b) site-2 protease (3B4R). (a) Rhomboids are serine proteases that utilise a catalytic dyad of serine and histidine (red) for hydrolysis of peptide bonds in the membrane. GlpG, a prokaryotic rhomboid from E. coli represents the core architecture with a bundle of six TM helices. A relatively short helix 4 and extended loop 3 (yellow) are placed at the centre of the molecule surrounded by other TM helices to form a hydrophilic cavity with numerous water molecules. Loop 5 caps this active site (orange). Loop 1, an essential component but whose function is unknown lies outside of the helical bundle (magenta). TM helices 1, 3 and 6 provide a structural support (green). Substrate is thought to enter through an opening between TM helices 2 and 5 (blue). (b) Site-2 protease from M. jannaschii is a zinc containing metalloprotease of unknown biological function. Zinc (red sphere) is coordinated by conserved histidines and aspartate (magenta). It is likely to be in a tetrahedral coordination with a water molecule (not observed due to limited resolution) forming the fourth coordination. TM helices 2, 3 and 4 (yellow) form the core that holds the key residues for catalysis. Discontinuous part of helix 4 is shown in brown. TM1 with a mixture of α-helix and β-strand is shown in blue. Outer helices TM 5 and 6 are in green.

Fig. 22

Membrane enzymes (a) thiol oxidase DsbB-DSbA (2ZUP), (b) leukotriene LTC₄ synthase trimer (2UUH) and (c) methane monooxygenase MMO (1YEW). Some enzymes are integral membrane proteins because they have hydrophobic substrates or because they are coupled to components of the electron transport chain. DsbB-DsbA complex: Disulphide bond formation in nascent polypeptides is catalyzed in the periplasm of Gram-negative bacteria by the highly reactive enzyme DsbA. DsbB is a left-handed four TM α-helical membrane protein that regenerates DsbA by reduction of a disulphide bond, followed by subsequent thiol oxidation in DsbB by ubiquinones. Conserved cysteine residues in DsbA (cys 30 and 33) and DsbB (cys 41, 44, 104, 130) play a crucial role in regeneration of DsbA by thiol exchange mechanism. It is possible to trap reaction intermediates of the DsbB-DsbA complex by mutating specific cysteine residues. The figure depicts one such intermediate where Cys³³ is mutated to alanine in DsbA and Cys¹³⁰ to serine in DsbB (both residues shown as green sticks). This results in an intramolecular disulphide bond (red) between Cys³⁰ of DsbA and Cys¹⁰⁴ of DsbB. Lack of cysteine at position 130 results in inter molecular disulphide between Cys⁴¹ and Cys⁴⁴ of DsbB (orange). Ubiquinone (brown) is found in a groove between TM1 and TM4 close to the Cys⁴¹ and Cys⁴⁴. When Cys33 of DsbA attacks the disulphide bond between DsbB-DsbA, it releases oxidised DsbA. Subsequent thiol exchange within DsbB restores its oxidised state. LTC₄ synthase: Membrane proteins involved in eicosanoid and glutathione metabolism are collectively called MAPEG (membrane associated proteins in eicosianid and glutathione metabolism). They play a key role in the generation of lipid mediators from arachidonic acid, which is responsible for bronchoconstriction leading to asthma. In addition, they play a major role in oxidative stress and xenobiotic detoxification. The membrane enzyme LTC₄ synthase converts leukotriene A4 to leukotriene C4 by covalent coupling to glutathione. Like other MAPEG family members, LTC₄ synthase is trimeric, with each monomer (cyan, blue and green) having four TM helices. Glutathione (red) binds at a crevice between two monomers. A detergent molecule (orange) that might mimic a substrate is shown close to glutathione. The structure of LTC₄ synthase suggests how glutathione is activated and coupled to the substrate but a definitive answers await more biochemical and probably more structural data. Methane monooxygenase: due to its inert nature, industrial oxidation of methane is an extremely difficult process. Methanotropic bacteria that use methane as their sole carbon source have evolved enzymes to carry out this difficult oxidation process. Membrane bound particulate methane monooxygenase is a homotrimer of three subunits giving a molecule with nine chains altogether (one monomer is shown in colour). Subunits A (dark blue) and C (yellow) together make up most of the TM domain with 12 (7 from subunit A and 5 from subunit C) helices. Subunit B (magenta) has two TM helices and a large soluble domain that contains two β-barrels. Three metal centres are observed in the crystal structure. A mononuclear copper (red) and a dinuclear copper centre (brown) are found in the soluble barrel domain of subunit B. A zinc ion (green) is found in the membrane coordinated by residues from subunit A and C. These metal centres must play a major role in activation of oxygen and subsequent generation of methanol.

Fig. 23

Architecture of porins (a) LamB trimer top view and (b) LamB monomer side view (1MAL). Found abundantly in the outer membrane of Gram-negative bacteria, β-barrel membrane proteins form the second major structural class. Depicted in the figure is a representative outer membrane porin LamB, a trimeric sugar transporter. Each monomer has an individual transport pathway and transports maltodextrin (red space filling model). LamB is an 18-stranded β-barrel with a pore that selects carbohydrates through interaction with aromatic amino acids. Other classical porins such as OmpF, OmpC, PhoE are also trimers but have 16 β-strands. All these proteins act as passive pores. The trimers of these proteins show remarkable stability even in presence of SDS.

Fig. 24

β-Barrel membrane proteins with diverse biological function (a) OmpLA (1QD5), (b) FepA (1FEP), (c) NalP (1UYN) and (d) VDAC (3EMN). β-Barrel membrane proteins are not just pore formers. They have evolved to act as receptors, enzymes and to transport specific molecules. OmpLA (a), is 12 stranded β-barrel that act as a phospholipase. OmpLA is active only as a dimer, this figure shows a monomer with its active site serine (red sticks) in complex with an inhibitor hexadecane sulphonyl fluoride (blue sticks). Transport of molecules such as iron and vitamin B₁₂ against a concentration gradient requires energy. Since the outer membrane is devoid of any proton motive force, energy is derived from the inner membrane by transient protein-protein interactions, called the TonB-dependent transport system. Outer membrane proteins that transport these substrates are typically 22-stranded β-barrels. FepA, an iron transporter, is an example (b) of a TonB-dependent transporter. The N-terminal TonB binding domain (purple) of this protein acts as a plug to prevent leakage. Substrate binding to the outer membrane protein results in a conformational change of the TonB binding domain that triggers binding of TonB and subsequent release of substrate. Encoded in a single polypeptide chain, autotransporters have a pore forming β-barrel and a passenger domain that is released to external medium by self-cleavage. NalP from Neisseria meningitis is one such protein (c) that shows the N-terminal passenger domain (red). β-barrel proteins are also found in the outer membranes of mitochondria and chloroplast. The structure of the voltage dependent anion channel (VDAC) from mitochondria reveals a 19-stranded β-barrel. Although, adjacent β-strands in trans-membrane proteins normally pair in antiparallel orientation, the first and last strands of VDAC are parallel, which is the first time this has been observed in a β-barrel membrane protein. An N-terminal helix (yellow) is found submerged halfway through the barrel. This helix has been implicated in voltage sensing, but the biological role of membrane potential in mitochondrial outer membranes and the orientation of VDAC is unclear.

Fig. 25

Magnesium transport proteins (a) pentameric CorA (2IUB) and (b) dimeric MgtE (2YVX). Side and periplasmic views of CorA: Pentameric CorA is made up of a large cytoplasmic domain and a TM domain with 10 helices, 2 from each monomer (brown). The stalk helix (yellow) that connects the two domains might play a role in transmitting a signal to the TM domain. Helices 5 (cyan) and 6 (pink) and the rest of cytoplasmic domain (green) acts as a divalent cation sensor (bound magnesium ions shown as red spheres). The C-terminal extension of TM2 domain that has a KKKKWL motif (blue) forms part of a basic sphincter ring. There is no bound Mg²⁺ in the pore and this structure probably represents a closed state. Side and periplasmic views of MgtE: In contrast to CorA, MgtE is a homodimer with 10 TM helices (brown), 5 from each monomer. The cytoplasmic domain consists of a superhelical N domain (green) and a cystathionine-β-synthase domain (cyan). The two plug helices (yellow) connect the TM and cytoplasmic domains. Magnesium ions (red spheres) are found at the interfaces between domains. One Mg²⁺ ion has been modelled in the pore near a conserved aspartate but due to moderate resolution the exact nature of the bound ion or the co-ordination geometry cannot be determined. Although different in architecture, the cytoplasmic domains of CorA and MgtE both act as divalent cation sensors. The loss of ion co-ordination due to lower intracellular Mg²⁺ concentration results in movement of the stalk helix in CorA or the connecting helix in MgtE, which transmits the signal to the TM domain. It is important to note that the bound cations in the cytoplasmic domains are at the interfaces of the various subdomains in both proteins. Except for a single aspartate in MgtE, hydrophobic residues line the pores of both proteins.

Fig. 26

Pore forming toxins a) ClyA (2WCD) b) α-HL (7AHL). (a) Side and top view of cytolysin A: in its membrane bound form, ClyA is a dodecameric α-helical pore-forming toxin. One subunit is highlighted to show the three-helix bundle. Helices B (green), C (yellow) and F (cyan) form the wall of the pore. Helix A (blue) and a short region of helix C comprise the membrane-inserted part. (b) Side and top view of α-hemolysin: α-HL is a β-barrel pore-forming toxin. The monomer of α-HL consists of the rim and amino latch domain (green) and a stem domain (blue) that forms the barrel. Each monomer contributes two β-strands to form a heptameric barrel.

Fig. 27

Some membrane protein structures are partially perturbed in detergent micelles. Most membrane proteins have been crystallised in the presence of detergent, which is not a native environment. There are a few examples where the presence of detergents and strenuous efforts in crystallisation have caused non-native structures to form well diffracting crystals. (a) The small multi-drug transporter EmrE normally exists as an antiparallel dimer in the membrane as well as in detergent micelles (Fig. 14 c). However, in one of the crystal forms obtained by 3D crystallisation, TM helices 1 to 3 from each monomer (blue and yellow) pack against each other to form a dimer, similar to that found in the native structure obtained in presence of substrate or in a lipid bilayer (Ubarretxena-Belandia et al. ; Chen et al. 2007; Fig. 14 c). However the TM 4 helices (red) are directed entirely away from the dimer core in all the monomers of the asymmetric unit. It is possible that the low pH used for crystallisation promotes disruption of the normal helix bundle and that contacts made by TM4 results in a well-ordered lattice. (b) Four independent molecules of β₁ adrenergic receptor are observed in one crystal form. In two of these molecules, the N-terminus of TM helix 1, representing about 10 amino acids, adopts a significantly different conformation from the structure of the two other molecules that represent the antagonist-bound state. The figure shows an overlay of molecules A and B (aquamarine), with the 60°-kinked conformation of the N-terminus of TM1 in molecule A coloured in red and the correct, non-kinked conformation of TM1 in molecule B in aquamarine. TM1 in GPCRs is the most distant helix from both the ligand binding and the G-protein binding sites and such distortion is certainly a result of crystallisation.

Fig. 28

Contributions from electron cryomicroscopy. (a) The structure of trimeric light-harvesting complex LHCII from pea (2W7B), determined by electron crystallography of 2D crystals, revealed a three-TM helical bundle and cofactors (chlorophyll in green and lutenes in salmon). (b) One example of how a low resolution 3D structure of a complete membrane protein complex, F₁F_o-ATPase from bovine mitochondria, determined by single particle electron cryomicroscopy, can help to create a framework for interpreting the substructures of different parts of the whole complex determined by X-ray crystallography or NMR spectroscopy. Figure from Dickson et al. (2006) with permission.

Fig. 29

NMR structures of membrane proteins (a) OmpX (1Q9F), (b) OmpG (2JQY), (c) diacyl glycerol (DAG) kinase (2KDC) and (d) phospholamban (1ZLL). All membrane protein structures so far determined by NMR of detergent solubilised proteins in solution are relatively small structures because the large size of the protein-detergent micelle complex broadens the NMR spectrum. Two representative NMR structures of the β-barrel membrane proteins OmpX, an eight-stranded β-barrel, and OmpG, a 14-stranded β-barrel are shown in (a) and (b), respectively. The largest β-barrel membrane protein structure determined by NMR is the mitochondrial porin VDAC (see section 9 and Fig. 24d). The structure of diacyl glycerol kinase (c) has so far been determined only by NMR spectroscopy. It reveals a trimer, each subunit having three TM helices that all make extensive intersubunit contacts. Phospholamban is a pentameric channel associated with the sarcoplasmic reticulum Ca²⁺-ATPase, SERCA. These two represent the α-helical membrane protein structures determined by NMR.