Most Transmembrane Proteins Have Membrane-Spanning α Helices

Soluble proteins exhibit hundreds of distinct localized folded structures, or motifs (see Figure 3-10). In comparison, the repertoire of folded structures in the transmembrane domains of integral membrane proteins is quite limited, with the hydrophobic α helix predominating. Proteins containing membrane-spanning α-helical domains are stably embedded in membranes because of energetically favorable hydrophobic and van der Waals interactions of the hydrophobic side chains in the domain with specific lipids and probably also because of ionic interactions with the polar head groups of the phospholipids.

A single α-helical domain is sufficient to incorporate an integral membrane protein into a membrane. However, many such proteins have more than one transmembrane α helix. Typically, a membrane-embedded α helix is composed of a continuous segment of 20–25 hydrophobic (uncharged) amino acids (see Figure 2-14). The predicted length of such an α helix (3.75 nm) is just sufficient to span the hydrocarbon core of a phospholipid bilayer. In many membrane proteins, these helices are perpendicular to the plane of the membrane, whereas in others, the helices traverse the membrane at an oblique angle. The hydrophobic side chains protrude outward from the helix and form van der Waals interactions with the fatty acyl chains in the bilayer. In contrast, the hydrophilic amide peptide bonds are in the interior of the α helix (see Figure 3-4); each carbonyl (C=O) group forms a hydrogen bond with the amide hydrogen atom of the amino acid four residues toward the C-terminus of the helix. These polar groups are shielded from the hydrophobic interior of the membrane.

To help you get a better sense of the structures of proteins with α-helical domains, we will briefly discuss four different kinds of such proteins: glycophorin A, G protein–coupled receptors, aquaporins (water/glycerol channels), and the T cell receptor for antigen.

Glycophorin A, the major protein in the erythrocyte plasma membrane, is a representative single-pass transmembrane protein, which contains only one membrane-spanning α helix (Figure 7-14a). The 23-residue membrane-spanning α helix is composed of amino acids with hydrophobic (uncharged) side chains, which interact with the fatty acyl chains in the surrounding bilayer. In cells, glycophorin A typically forms dimers: the transmembrane helix of one glycophorin A polypeptide associates with the corresponding transmembrane helix in a second glycophorin A to form a coiled-coil structure (Figure 7-14b). Such interactions of membrane-spanning α helices are a common mechanism for creating dimeric membrane proteins, and many membrane proteins form oligomers (two or more polypeptides bound together noncovalently) by interactions between their membrane-spanning helices.

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FIGURE 7-14 Structure of glycophorin A, a typical single-pass transmembrane protein. (a) Diagram of dimeric glycophorin, showing its major sequence features and its relation to the membrane. The single 23-residue membrane-spanning α helix in each monomer is composed of amino acids with hydrophobic (uncharged) side chains (red and green spheres). By binding negatively charged phospholipid head groups, the positively charged arginine and lysine residues (blue spheres) near the cytosolic side of the helix help anchor glycophorin in the membrane. Both the extracellular and the cytosolic domains are rich in charged residues and polar uncharged residues; the extracellular domain is heavily glycosylated, with carbohydrate chains (green diamonds) attached to specific serine, threonine, and asparagine residues. (b) Molecular model of the transmembrane domain of dimeric glycophorin A corresponding to residues 73–96. The hydrophobic side chains of the α helix in one monomer are shown in pink; those of the other monomer, in green. Residues depicted as space-filling structures participate in van der Waals interactions that stabilize the coiled-coil dimer. Note how the hydrophobic side chains project outward from the helix, toward what would be the surrounding fatty acyl chains.
[Part (b) data from K. R. MacKenzie et al., 1997, Science 276:131, PDB ID 1afo.]

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A large and important group of integral membrane proteins is defined by the presence of seven membrane-spanning α helices. This group includes the large family of G protein–coupled cell-surface receptors discussed in Chapter 15, many of which have been crystallized. One such multipass transmembrane protein of known structure is bacteriorhodopsin, a protein found in the membranes of certain photosynthetic bacteria; it illustrates the general structure of all these proteins (Figure 7-15a). Absorption of light by the retinal group covalently attached to this protein causes a conformational change in the protein that results in the pumping of protons from the cytosol across the bacterial membrane to the extracellular space. The proton concentration gradient thus generated across the membrane is used to synthesize ATP during photosynthesis (see Chapter 12). In the high-resolution structure of bacteriorhodopsin, the positions of all the individual amino acids, the retinal group, and the surrounding lipids are clearly defined. As might be expected, virtually all the amino acids on the exterior of the membrane-spanning segments of bacteriorhodopsin are hydrophobic, permitting energetically favorable interactions with the hydrocarbon core of the surrounding lipid bilayer.

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FIGURE 7-15 Structural models of two multipass membrane proteins. (a) Bacteriorhodopsin, a photoreceptor in certain bacteria. The seven hydrophobic α helices in bacteriorhodopsin traverse the lipid bilayer roughly perpendicular to the plane of the membrane. A retinal molecule (black) covalently attached to one helix absorbs light. The large class of G protein–coupled receptors in eukaryotic cells also has seven membrane-spanning α helices; their three-dimensional structure is thought to be similar to that of bacteriorhodopsin. (b) Two views of the glycerol channel Glpf, rotated 180° with respect to each other along an axis perpendicular to the plane of the membrane. Note the several membrane-spanning α helices that are at oblique angles, the two helices that penetrate only halfway through the membrane (purple with yellow arrows), and the one long membrane-spanning helix with a “break” or distortion in the middle (purple with yellow line). The glycerol molecule in the hydrophilic “core” is colored red. The protein structure was approximately positioned in the hydrocarbon core of the membrane by finding the most hydrophobic 3-µm slab of the protein perpendicular to the membrane plane.
[Part (a) data from H. Luecke et al., 1999, J. Mol. Biol. 291:899. Part (b) data from J. Bowie, 2005, Nature 438:581–589, PDB ID 1c3w and D. Fu et al., 2000, Science 290:481–486, PDB ID 1fx8.]

The aquaporins are a large family of highly conserved proteins that transport water, glycerol, and other hydrophilic molecules across biomembranes. They illustrate several aspects of the structure of multipass transmembrane proteins. Aquaporins are tetramers of four identical subunits. Each of the four subunits has six membrane-spanning α helices, some of which traverse the membrane at oblique angles rather than perpendicularly. Because all aquaporins have similar structures, we will focus on one, the glycerol channel Glpf, whose structure has been especially well defined by x-ray diffraction studies (Figure 7-15b). This aquaporin has one long transmembrane helix with a bend in the middle, and more strikingly, there are two α helices that penetrate only halfway through the membrane. The N-termini of these helices face each other (yellow Ns in the figure), and together they span the membrane at an oblique angle. Thus some membrane-embedded helices—and other, nonhelical, structures we will encounter later—do not traverse the entire bilayer. As we will see in Chapter 11, these short helices in aquaporins form part of the glycerol/water-selective pore in the middle of each subunit. This structure highlights the considerable diversity in the ways membrane-spanning α helices interact with the lipid bilayer and with other segments of the protein.

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FIGURE 7-16 Annular phospholipids. Side view of the three-dimensional structure of one subunit of the lens-specific aquaporin 0 homotetramer, crystallized in the presence of the phospholipid dimyristoylphosphatidylcholine, a phospholipid with 14 carbon-saturated fatty acyl chains. Note the lipid molecules forming a bilayer shell around the protein. The protein is shown as a surface plot (the lighter background molecule). The lipid molecules are shown in space-filling format; the polar lipid head groups (gray and red) and the lipid fatty acyl chains (the extended black and gray structures) form a bilayer with almost uniform thickness around the protein. Presumably, in the membrane, lipid fatty acyl chains cover the whole of the hydrophobic surface of the protein; only the most ordered of the lipid molecules would be resolved in the crystallographic structure.
[Data from A. Lee, 2005, Nature 438:569–570, and T. Gonen et al., 2005, Nature 438:633–688, PDB ID 2b6o.]

The specificity of phospholipid-protein interactions is evident from the structure of a different aquaporin, aquaporin 0 (Figure 7-16). Aquaporin 0 is the most abundant protein in the plasma membrane of the fiber cells that make up the bulk of the lens of the mammalian eye. Like other aquaporins, it is a tetramer of identical subunits. The protein’s surface is not covered by a set of uniform binding sites for phospholipid molecules. Instead, fatty acyl chains pack tightly against the irregular hydrophobic outer surface of the protein. The lipids involved in this interaction are referred to as annular phospholipids because they form a tight ring (annulus) of lipids around the protein that are not easily exchanged with bulk phospholipids in the bilayer. Some of the fatty acyl chains are straight, in the trans conformation (see Chapter 2), whereas others are kinked in order to interact with bulky hydrophilic side chains on the surface of the protein. Some of the lipid head groups are parallel to the surface of the membrane, as is the case in pure phospholipid bilayers. Others, however, are oriented almost at right angles to the plane of the membrane. Thus there can be specific interactions between phospholipids and membrane-spanning proteins, and the function of many membrane proteins can be affected by the specific types of phospholipids present in the bilayer.

In addition to the predominantly hydrophobic (uncharged) residues that serve to embed integral membrane proteins in the bilayer, many α-helical transmembrane segments contain polar or charged residues. Their amino acid side chains can be used to guide the assembly and stabilization of multimeric membrane proteins. The T cell receptor for antigen is a case in point: it is composed of four separate dimers, the interactions of which are driven by charge-charge interactions between α helices at the appropriate “depth” in the hydrocarbon core of the lipid bilayer (Figure 7-17). The electrostatic attraction of positive and negative charges on each dimer helps the dimers to “find each other.” Thus charged residues in otherwise hydrophobic transmembrane segments can help guide assembly of multimeric membrane proteins.

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FIGURE 7-17 Charged residues can orchestrate the assembly of multimeric membrane proteins. The T cell receptor (TCR) for antigen is composed of four separate dimers: an αβ pair directly responsible for antigen recognition, and accessory subunits collectively referred to as the CD3 complex. These accessory subunits include the γ, δ, ε, and ζ subunits. The ζ subunits form a disulfide-linked homodimer. The γ and δ subunits occur in complex with an ε subunit, to generate a γε and a δε pair. The transmembrane segments of the TCR α and β chains each contain positively charged residues (blue). These residues allow recruitment of corresponding δε and γε heterodimers, which carry negative charges (red) at the appropriate depth in the hydrophobic core of the bilayer. The ζ homodimer docks onto the charges in the TCR α chain (dark green), while the γε and δε subunit pairs find their corresponding partners deeper down in the hydrophobic core on both the TCR α and TCR β chain (light green). Charged residues in otherwise nonpolar transmembrane segments can thus guide assembly of higher-order structures.
[Data from K. W. Wucherpfennig et al., 2010, Cold Spring Harb. Perspect. Biol., 2:a005140, PDB ID 1xmw; M. E. Call et al., 2006, Cell, 127:355, PDB ID 2hac; and L. Kjer-Nielsen et al., 2003, Immunity, 18:53, PDB ID 1mi5.]