Membrane proteins - module I


What membrane proteins look like

This section is largely based on:
von Heijne, G. (1997) Principles of membrane protein assembly and structure. Progr.Biophys.Mol.Biol., 66:113-139.
von Heijne, G. (1999) Recent advances in the understanding of membrane protein assembly and structure. Quart.Rev.Biophys., 32:285-307.


Membrane proteins come in a variety of sizes and shapes, though the basic architectural principles apparent from the available 3D structures are far less diverse than those of the globular proteins. This is a consequence of the requirement that membrane proteins either must bind to one leaflet of, or completely span a lipid bilayer while at the same time maintaining contact with the surrounding aqueous medium. Peripheral binding to one side of the bilayer can be mediated by surface-exposed residues protruding into one leaflet of the bilayer, and membrane-spanning structures can be built either from beta-barrels or from bundles of transmembrane alpha-helices. Large extra-membraneous domains do not seem to be much influenced by their attachment to the bilayer, and can essentially be viewed as tethered globular proteins.

The structural differences between membrane and globular proteins do not only result from the different physico-chemical requirements imposed by their different environments, but also reflect fundamental differences in the mechanisms responsible for their folding and assembly. While globular proteins encounter only one kind of environment from the time of their synthesis until they are degraded in old age, membrane proteins are first exposed to aqueous surroundings and only later gain access to the milieu for which they are designed. The question of how the controlled integration of a membrane protein into the lipid bilayer takes place is still not fully worked out, and there are certainly aspects of membrane protein structure that will probably only be fully appreciated once this has been accomplished.

Three basic structures: helix bundles, beta-barrels, and peripheral proteins

Two major factors determine the structure of proteins spanning a lipid bilayer: the high free energy cost of burying charged moieties in the hydrophobic environment, and the necessity to maintain hydrogen bonds to all backbone peptide groups. The first factor essentially means that lipid-exposed residues tend to be hydrophobic (A, L, I, M, V, F), while the second factor means that transmembrane polypeptide segments tend to fold either into alpha-helices or into closed beta-barrels. It is thus no big surprise that the known 3D structures of integral membrane proteins are either bundles of helices or beta-barrels.

A third class of membrane proteins are those that are peripherally bound to only one side of a membrane. The membrane-binding surfaces in such proteins have no conserved structure, but are rich in hydrophobic and positively charged residues, presumably mediating interactions both with negatively charged phospholipid headgroups and with the more non-polar environment deeper down in the bilayer.

Some representative membrane protein structures

While the number of known membrane protein 3D structures is still limited, the general features discussed above are nevertheless readily apparent. Here are some typical membrane proteins (note: you need to have a program like RasMol or Swiss PDB Viewer installed to look at the PDB files):

Bacteriorhodopsin: Bacteriorhodopsin, a light-driven proton pump from Halobacterium halobium, was the first integral membrane protein to be shown to consist of a bundle of transmembrane helices oriented roughly perpendicular to the membrane plane (Henderson and Unwin, 1975). 15 years later, the structure had been determined to sufficient resolution to allow the bulky aromatic side chains of the transmembrane helices to be built into the electron density (PDB 1BRD), and another 6 years of data collection and analysis finally produced a density map where most side chains, both in the helices and the connecting loops, could be fitted (PDB 2BRD). More recently, bacteriorhodopsin structures representing different steps in the photocycle have been solved to very high resolution by X-ray crystallography (e.g., PDB 1C3W). Bacteriorhodopsin is not only one of the best structurally and functionally characterized integral membrane proteins, but has also served as the test-bed for the development of electron crystallography and, more recently, for the development of so-called cubic lipid phases for growing 3D crystals.

Bacteriorhodopsin (PDB 2BRD). The light-absorbing retinal is in white and lipid molecules are shown as stick models.
The transmembrane helices in bacteriorhodopsin are composed mainly of hydrophobic residues. A few charged residues are buried within the structure, serving important functions in the proton-relay system. Bacteriorhodopsin has been widely used as a starting structure for modeling many medically important G-protein coupled receptors, though with the publication of the 3D structure of rhodopsin, a mammalian G-protein coupled light receptor (PDB 1F88), these models have become largely obsolete.
R hodopsin (PDB 1F88). The light-absorbing retinal is shown in white.


The photosynthetic reaction center: The structure of the photosynthetic reaction center from Rhodopseudomonas viridis was solved by X-ray crystallography in 1985 (PDB 1PRC), followed 2 years later by the closely related one from Rhodobacter sphaeroides (PDB 1AIG). The structure is considerably larger than bacteriorhodopsin, with a total of three membrane-bound and one peripheral protein subunits, 11 transmembrane helices, four bacteriochlorophylls, two bacteriopheophytins, two quinones, and a nonheme iron. The heart of the protein is the electron-conducting chain of co-factors held in place by the protein scaffold (look at this in RasMol by showing the hetero atoms in spacefill and the protein in backbone representation).

The  photosynthetic reaction center (PDB 1PRC). The elctron-conducting cofactors are shown in white. The membrane-embedded domain is composed of the helix bundle in the lower part of the picture.

Since this was the first high resolution structure of a membrane protein available, it provided the first clear indications of how transmembrane helices pack. The atomic packing density in the interior of the protein is similar to what is found in globular proteins, as is the average hydrophobicity of the internal residues. Residues exposed to the lipid environment are more hydrophobic than the internal ones, just the opposite of what is the case for globular proteins. The exposed surface residues are more variable between homologous proteins from different species than are the internal residues (Rees et al., 1989).

Many of the transmembrane helices protrude quite a distance from the bilayer, and consequently have a central hydrophobic part flanked by more polar residues. Interestingly, Tyr and Trp residues tend to be concentrated near the presumed position of the lipid headgroup region (verify this by highlighting W and Y in the structure!).

Cytochrome c oxidase: The bacterial complex contains three central subunits, while the mitochondrial one has no less than 13 subunits, 10 of which have one or more transmembrane helix, pushing the total number of helices to 28 (PDB 1OCC). In addition, the two catalytically important Cu-sites, two hemes, and 8 phospholipids have been located in the structure.

Analysis of the structure of the mitochondrial oxidase has suggested a rather detailed consensus structure that seems to be shared between the cytochrome c oxidase, the bacterial photosynthetic reaction center, and bacteriorhodopsin, and that may thus be generally valid for helix bundle membrane proteins (Elofsson et al., 1996): a central, helical region of about 20 Å rich in aliphatic residues and Phe, an "aromatic belt" composed of Trp and Tyr similar to but less prominent than that seen in the beta-barrel porin structures (see next section), a region near the helix ends with amidated and charged residues, and helix breaking Pro residues just outside the helices (verify by using RasMol!).

Cytochrome c oxidase (PDB 1OCC). Cofactors are shown in white.


The KcsA potassium channel: A major breakthrough in the membrane protein structure field was the determination of the structure of the tetrameric K+ channel KcsA from Streptomyces lividans (PDB 1BL8). The KcsA channel is thought to be similar in overall architecture to a wide range of K+ channels, both from prokaryotes and eukaryotes.

The KcsA potassium channel. The white loops protruding inbetween the transmembrane helices form the ion selectivity filter.

The most interesting part of the KcsA channel is the so-called selectivity filter region that is formed by a short strand-turn-helix structure that dips into the channel from the periplasmic side. Backbone oxygens from this so-called P-loop region form a constricted passage, just wide enough to efficiently desolvate K+ ions but too wide to desolvate smaller Na+ ions. As a result of this, the permeability for K+ ions is at least 4 orders of magnitude larger than for Na+ ions. Other ion binding sites in the channel also contribute to the overall permeation rate (Roux and MacKinnon, 1999).

KcsA viewed from above. The tiny black hole in the middle is the entrance to the ion channel. Oxygens (in red) lining the hole strip waters from the hydrated K+ ion.

The MscL pressure-sensitive channel: MscL is a bacterial, non-selective, mechanosensitive ion channel. It is composed of five identical subunits, each contributing two transmembrane helices (PDB 1MSL). The pore structure extends into the cytoplasm, where five helices, one from each of subunit, form a bundle closely apposed to the transmembrane helices. The pore is lined by mainly polar amino acids, and has a hydrophobic occlusion near the cytoplasmic side of the membrane thought to form the gate. It has been speculated that increased lateral tension in the membrane will pull the transmembrane helices away from each other, thus opening the gate.

The McsL ion channel (PDB 1MSL). The membrane-embedded domain is formed by the upper helix bundle.

Aquaporin: Aquaporins are water channels that exclude ions. Aquaporins are found in essentially all organisms, and have major biological and medical importance. The structure of an aquaporin from human erythrocytes has been solved by electron crystallography to 3.8 Å resolution (PDB 1FQY). The protein has a pseudo-symmetric right-handed helix bundle structure, with the first three transmembrane helices having significant similarity in sequence and structure to the last three. The most interesting aspect of the aquaporin structure is the water selectivity filter located in the middle of the membrane-spanning domain. Thanks to a judicious placement of hydrogen-bonding groups in the channel wall, a water molecule in transit needs only lose one H-bond when it traverses the filter. Ions, on the other hand, have no way to shed their co-ordinated waters and hence are excluded from the channel.

Model for water permeation through aquaporin 1 (Nature 407:599). Note how each water molecule can maintain most of its H-bonds throughout the passage. The central water molecule does not form H-bonds to its neighbors (bottom right), thus interrupting the H-bond chain through the pore and preventing the formation of a 'proton wire' that would make the pore leaky to protons.

Beta-barrel proteins: Beta-barrel proteins are typically found in the outer membrane of Gram-negative bacteria. They are formed by large anti-parallel beta-barrel structures with short loops facing the periplasm and larger loops protruding outside the outer membrane (Koebnik et al., 2000). For porins -which form passive diffusion pores - substrate specificity and translocation kinetics depend on a segment of the chain that loops into the barrel.

Not surprisingly, the outer surface of the barrel is composed of hydrophobic residues, while residues on the inside may be both hydrophobic and hydrophilic. Since the membrane-spanning segments of the chain are beta-strands, this means that only every second residue is facing the lipids and hence needs to be hydrophobic. The strands in the porin from R. capsulatus are tilted by about 30- 60 degrees relative to the membrane normal, and are 6-17 residues long. The long hydrophobic segments typical of the helix bundle membrane proteins are absent from the porins, and in fact such segments, if introduced by molecular genetic techniques into an outer membrane protein, become trapped in the inner membrane during translocation, thus preventing assembly into the outer membrane.

OmpA (PDB 1BXW). The OmpA beta-barrel has 8 strands, and is not wide enough to form a pore in the membrane.
A notable feature of the porin structures are the belts of aromatic residues that point out towards the lipid headgroup region on either side of the bilayer. The functional significance of these aromatic belts is not known, though it has been speculated that they may serve to keep the molecule in a fixed orientation relative to the bilayer. As noted above, a similar clustering of aromatic residues near the lipid headgroup region, Trp and Tyr in particular, is seen also in the helix bundles proteins.
The MalE maltose-selective porin (PDB 1MAL). The beta-barrel has 16 strands. Trp and Tyr residues are shown in spacefill; note the 'aromatic belts' flanking the membrane-spanning domain.

The beta-barrels can be expanded to a size that allows entire folded domains of the protein to insert into the middle of the pore, as seen in the FhuA siderophore-transporter. In this way, non-hydrophobic protein domains can be located in the middle of a membrane, shielded from contact with the surrounding lipid.

The FhuA siderophore transporter (PDB 1BY3). The beta-barrel has 22 strands (?). The non-hydrophobic 'plug' domain is seen in magenta and white in the middle of the molecule.


Prostaglandin H2 synthase: Prostaglandin H2 synthase binds to the lumenal leaflet of the endoplasmic reticulum membrane, and is thus classified as a peripheral membrane protein. In contrast to the peripherally bound globular subunits of, e.g., the photosynthetic reaction center and cytochrome c oxidase, its membrane binding is not mediated by other proteins but is the result of direct protein-lipid interactions.

The structure of prostaglandin synthase strongly suggests that binding to the bilayer is through four amphiphilic alpha-helices which, in contrast to most helices in globular proteins, has a series of solvent-exposed hydrophobic residues flanked by basic residues. These hydrophobic residues are postulated to insert into the bilayer, while the flanking charged residues may interact with the phospholipid headgroups. The amphiphilic helices are thus thought to lie more or less flat on the membrane surface, a mode of binding that has also been seen in some small lytic peptides such as magainin (Bechinger et al., 1993).

Prostaglandin H2 synthase (PDB 1PRH). The membrane-binding part is formed by the alpha-helices sticking out from bottom of the molecule.


© Gunnar von Heijne, Stockholm Bioinformatics Center, 2000