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.
Background
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).
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.
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.OmpA (PDB 1BXW). The OmpA beta-barrel has 8 strands, and is not wide enough to form a pore in the membrane.
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
