Protein-Protein Interactions in the Membrane: Sequence, Structural, and Biological Motifs (original) (raw)

. Author manuscript; available in PMC: 2013 Sep 12.

Published in final edited form as: Structure. 2008 Jul;16(7):991–1001. doi: 10.1016/j.str.2008.05.007

Abstract

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.

Introduction

Helical integral membrane proteins are involved in diverse biological processes, from the fusion of sperm and egg at conception to apoptotic events during cell death. The rapidly expanding list of polytopic membrane protein structures has provided insight into how folding and packing of their transmembrane (TM) segments contribute to their diverse functions. More recently, we have begun to understand the importance of single-span TM helices beyond being anchors for more “interesting” water-soluble domains or mere models for multipass protein folding. Like small modular domains, such as leucine zippers that mediate protein complex formation in water-soluble proteins, TMs can direct protein-protein interactions within the membrane (Bechinger, 2000; Call and Wucherpfennig, 2005; Curran and Engelman, 2003; MacKenzie, 2006; Senes et al., 2004) and participate in signal transduction across lipid bilayers (Klein-Seetharaman, 2005).

Here, we will discuss sequence and geometric motifs that are important for interactions between single-pass TM helices, with a special focus on biological function. In addition to organizing these interactions by sequence and structure, they can be broken down into two broad functional categories (Figure 1): (1) relatively static, kinetically stable contacts between TM helices that are important for the assembly of multiprotein complexes; and (2) regulated-switchable interactions involved in signal transduction across bilayers via changes in oligomeric state and/or conformation. Thermodynamically, these groupings are likely to exist in a continuum and be affected by interactions within the water-soluble compartment. However, biologically (and medically) these differences can be significant, because a number of diseases result from changes in these equilibria.

Figure 1. Two General Categories of TM Interactions.

Figure 1

(A) Interacting TM helices can form static associations in the membrane or (B) participate in regulated switchable interactions in a dynamic equilibrium that regulates signaling. A simplified schematic of the coupled equilibria involving a dimeric receptor-ligand complex is depicted; however, signal transduction can potentially travel in both directions across the membrane. The receptor is in equilibrium between the monomeric and dimeric states, and ligand can bind to either. Along the bottom path, ligand binding to a dimeric receptor leads to a conformational change and signal transduction across the membrane. On the top path, the binding of ligand to a monomeric receptor can induce dimerization of the receptor and the subsequent conformational changes. Depending on the local ligand concentration and the expression level of the receptor in the membrane, different cell types and tissues can potentially fine-tune their responses as needed.

We will also discuss a special subclass of TM helices that function as modulators of other TM proteins. Examples of this class of proteins include channel-regulating peptides, such as phos-pholamban, as well as viral proteins that activate growth factor receptors. The surprising sequence specificity of these natural peptides and recent computational methods for the design of novel TM regulating agents point toward potential therapeutic avenues for the targeting of TM interactions.

Geometric Motifs in Multipass Membrane Proteins

What are the geometric characteristics of helix-helix interactions in membrane proteins (Chamberlain et al., 2003) and how does an amino acid sequence predispose the helices to adopt these geometries? Early work focused on distributions of the interhelical angles and distances and on the composition of the side chains packed at the interface. These studies have shown that helices tend to approach somewhat more closely in membrane-soluble proteins than in water-soluble proteins, although the distributions tend to be broad and overlapping (Bowie, 1997; Gimpelev et al., 2004). Distributions of interhelical angles also overlap, but the extent to which this reflects geometric preferences, versus biases associated with random distributions of two vectors, is not clear (Bowie, 1997). Another difficulty with early statistical surveys is that they considered only two parameters (interhelical distance and crossing angle). Six parameters (three Eulerian angles and three distances) are required to define the mutual orientation of two helices (Engel and DeGrado, 2005); more would be required for nonideal helices.

An alternate approach (Figure 2) involves clustering interacting helical pairs according to their three-dimensional similarity (Gimpelev et al., 2004; Walters and DeGrado, 2006). Surprisingly, two-thirds of the helical pairs selected from high-resolution crystal structures of membrane proteins fell within just four clusters, in which Cα atoms from each helix in the pair were within 1.5 Å rmsd of the centroid structure (Walters and DeGrado, 2006). The largest cluster, which comprises 29% of the library, consists of antiparallel helices with left-handed crossing angles of approximately 20° and packing of amino acids with small side-chains that are spaced every seven residues in the sequence. Because Gly, Ala, and Ser (G, A, S) are frequently found at this helix-helix interface, the cluster was designated the antiparallel-GASLeft motif. Right-handed motifs with approximately 40° crossing angles show a similar tendency to segregate small residues to the helix-helix interface, but spaced at four-residue intervals leading to the designation of parallel and antiparallel GASRight motifs.

Figure 2. The Majority of Interacting TM Pairs Use a Limited Number of Structural Motifs.

Figure 2

(A) Geometric clustering of interacting TM pairs in the crystallographic database by rmsd places two-thirds of TM helices into four categories: parallel or antiparallel GASLeft, and parallel or antiparallel GASRight (Walters and DeGrado, 2006).

(B) The majority of known interacting monotypic TMs fall into parallel clusters. Parallel GASRight pairs have a ~40° crossing angle and a GX3G-like motif. Parallel GASLeft helices have a ~20° crossing angle and often a heptad repeat of small residues (G/A/S).

It is important to note that kinked structures can be accommodated adjacent to or within many of these motifs, and they might also establish unique packing motifs and hydrogen-bonding networks because of unpaired backbone carbonyls (Senes et al., 2004). Proline residues, which could introduce a bend in an alpha helix are common in membrane proteins (Senes et al., 2000), are overrepresented in interacting TM libraries (Dawson et al., 2002) and have been demonstrated to be critical for membrane protein function (Yohannan et al., 2004).

Sequence Motifs in Single-Pass Membrane Proteins

To date, most interactions between single-span TM helices involve parallel helix packing. For homomeric bundles, this is a consequence of all known single-span proteins being unidirectionally inserted into membranes. In the case of heteromeric interactions, the individual proteins are often evolutionarily related and share a common orientation leading to parallel bundles when their TM helices interact.

Right-Hand-Parallel Helix Packing Motifs

The core geometric and sequence elements of the GASRight motif were first recognized by Engelman and coworkers, who demonstrated that a GX3G sequence in the glycophorin A (GpA) TM domain was necessary for homodimerization and adopted an approximately 40° right-handed crossing angle (Lemmon et al., 1992; MacKenzie et al., 1997; Senes et al., 2000). This motif has since been observed in water-soluble proteins (Kleiger et al., 2002). In the classic GpA GX3G motif, the small Gly residues create a shallow groove that complements the surface of a second helix. The association is stabilized by van der Waals interactions resulting from the excellent geometric fit and also by weak hydrogen bonding between CαH groups on one helix and the carbonyls of a neighboring helix (Senes et al., 2001). Besides the original GpA NMR structure (MacKenzie et al., 1997), there have been recent studies of GX3G containing TM homodimers (Bocharov et al., 2007, 2008), including the structure of the Ff bacteriophage major coat protein, which adopts a noncanonical 27° crossing angle (Wu et al., 2007).

Extensive mutagenesis of the GpA TM has shown that the sequence L75IxxGVxxGVxxT87 is most important for dimerization in SDS-micelles (Lemmon et al., 1992). Subsequent studies by analytical ultracetrifugation and in bacterial reporter systems have largely supported this motif, with some differences between micellar and membrane systems, depending on the hydrophobicity of the mutations (Duong et al., 2007). It is interesting to note the presence of beta-branched amino acids—Ile, Val, and Thr—which have fewer low-energy rotameric conformations and allow helix dimerization to occur at minimal entropic cost (MacKenzie et al., 1997). Recently, it also has been shown that phenylalanine can reinforce GX3G dimerization (Unterreitmeier et al., 2007). Consistent with the GASRight motif found in multi-pass proteins, the small residues Ala and Ser can replace one or both of the Gly residues (Senes et al., 2000, 2004; Walters and DeGrado, 2006), or small amino acids can occur repetitively every four residues to create extended “glycine zippers” (Kim et al., 2005).

Left-Hand-Parallel Helix Packing Motifs

TM interactions consistent with GASLeft packing are also very common in single-pass proteins. Like water-soluble coiled coils (O’Shea et al., 1991), they often have 20° crossing angles and heptad sequence repeats (Langosch and Heringa, 1998; Walters and DeGrado, 2006). For example, the class II MHC α– and β-chains contain heptad repeats of Gly residues at three consecutive “a” positions, with the remaining “a” position being filled by a small residue (Cys or Ser) (Cosson and Bonifacino, 1992). Mutation to many of the Gly residues disrupts the formation of heterodimers between the two chains (Cosson and Bonifacino, 1992). Similarly, Gly residues fill several of the “d” positions. Synthetic peptides containing a similar heptad repeat associate to form dimers in micelles (Lear et al., 2004).

graphic file with name nihms513265u1.jpg

Heptad repeats with Ser at the “a” position have also been found in a number of other structures. Such motifs have been used in the design of ion channel peptides (Lear et al., 1988) and drive dimerization of the erythropoietin receptor TM (Ebie and Fleming, 2007; Kubatzky et al., 2001; Ruan et al., 2004a). Adamian and Liang (2002) have also described an antiparallel “serine zipper” motif in which interacting Ser sidechains are spaced at seven-residue increments in the crystal structures of many transmembrane proteins. Model peptides with a SaxxLdxxx repeat were found to associate to form both parallel and antiparallel dimers by equilibrium thiol-disulfide exchange (North et al., 2006). A variant containing an AaxxLdxxx repeat dimerized with similar affinity, indicating that the Ser might form intrachain H-bonds with the carbonyl at position i-4 and that its small size was the dominant factor for oligomerization (North et al., 2006). By use of a genetic selection from a randomized library of TM sequences, Engelman and coworkers also demonstrated that SxxSSxxT and SxxxSSxxT motifs could strongly drive dimerization in bacterial membranes. Such motifs are potentially consistent with either GASRight or GASLeft (Dawson et al., 2002).

Compared with optimized GX3G motifs in the GASRight family, the stabilities of the left-handed dimers discussed in this section are frequently rather weak. However, as we will see in the next section, hydrogen bonds between more polar groups, such as Asp, Asn, Gln, or Glu, can work in concert with these packing interactions to strongly stabilize left-handed motifs.

Polar Motifs

Strongly polar side chains can play an important role in the association of TM helices in a variety of packing motifs. In some cases, they help to augment or provide geometric specificity to an energetically dominant folding motif. For example, a Thr residue at the helix-helix interface of GpA appears to cooperate with the GX3G motif to increase the affinity and specificity of the inter-action (Duong et al., 2007; Lemmon et al., 1992). A recent NMR structure of the BNIP3 TM homodimer found glycophorin-like packing between small amino acids, as well as extensive hydrogen bonding between Ser and His residues (Bocharov et al., 2007). This structure was consistent with previous mutagenesis and computational studies (Metcalf et al., 2007; Sulistijo and MacKenzie, 2006).

In other cases, a single polar residue can play a dominant role in stabilizing helix-helix associations. Model peptides containing a single Asn, Asp, Gln, or Glu dimerize and/or trimerize in detergents, liposomes (Choma et al., 2000; Gratkowski et al., 2001; Zhou et al., 2000, 2001), and bacterial membranes (Dawson et al., 2003; Ruan et al., 2004b). Oligomerization of an Asn-containing peptide was increased by adding a second Asn at position i+7 (Lear et al., 2003) or an adjacent Ser or Thr residue to form a hydrogen-bonded network (Tatko et al., 2006).

The solution structure of the TM helix of the T cell receptor (TCR) ζζ-chain dimer (Figure 3) nicely demonstrates how H-bonding can establish a left-handed homodimer (Call et al., 2006). Previous studies showed that homodimerization was dependent on an N-terminal interchain disulfide bond and a conserved Asp residue at position i+4 relative to the Cys (Call et al., 2002). The nature of this residue was found to be extremely important, because a Glu mutation maintained the homodimer, whereas Asn, Ser, and Ala mutations reduced dimerization by 60%, 60%, and >80%, respectively. Mixed heterodimers Asp/ Glu, Asp/Asn, and Asp/Ser all formed equally well, whereas Asp/Ala dimerization was reduced by nearly 40%. On the basis of NOEs with water and the mutagenesis data, it was proposed that the two aspartic acids form an extensive hydrogen-bonding network with at least one water molecule, the cysteine carbonyls, and aspartic acid backbone amides. Although the protonation state of the Asp is not yet known, the carboxylates on neighboring chains are in very close proximity (~2.5 Å oxygen-to-oxygen distance) in the computed structure, suggesting that they might form a carboxylate-carboxyl pair with a single proton shared between the two carboxylates (Jasti et al., 2007; Wohlfahrt, 2005).

Figure 3. The TCR-ζ Chain Has a Geometric Motif Similar to the Leucine Zipper GCN4-P1.

Figure 3

(A) Superposition of the TCR-ζ chain TM dimer (Call et al., 2006) and the GCN4 backbones (O’Shea et al., 1991).

(B) Schematic of the crucial interactions within the TCR-ζ helical dimer.

(C) TCR-ζ chain interactions including a Cys-disulfide, Asp-Asp hydrogen bonding, and Thr-Tyr hydrogen bonding.

(D) The analogous GCN4-P1 interactions, including the Asn-Asn and Lys-Glu pairs.

The NMR structure of the ζζ-dimer has a left-handed crossing angle of 20°, which is quite similar to the value seen in left-handed coiled coil peptides. Indeed, the structure superimposes quite well onto the structure of GCN4-P1 (Figure 3A), a two-stranded coiled coil from the yeast transcription factor GCN4. GCN4-P1 and the ζ-chain contain analogous heptads—NxxLxxx and DxxLxxx (Figure 3B), respectively. Although water-soluble coiled-coils often have salt bridges between positions “g” and “e” of successive heptads, a hydrogen bond is formed between Tyr and Thr residues in the ζ-chain, as can be seen by comparing Figure 3C with Figure 3D. Thus, the same basic framework is retained in both cases, but different interactions are used to stabilize the membrane-soluble versus the water-soluble structures.

Biological Significance

The geometric and sequence motifs described above can participate in two broad categories of interactions. In the first, the TMs form relatively static contacts that are necessary for the assembly of a functional protein complex and for proper folding and export from endoplasmic reticulum. In the second, TMs undergo dynamic conformational changes important for signaling; this process can involve a change in association state and/or lateral, vertical, and rotational motions in the membrane (Qiu et al., 2006). We refer to this class of TMs as “switchable.” Switchable TM interactions may not be the dominant force regulating protein-protein interactions, but rather fine-tune the system’s energetics. The biological importance of these interactions is highlighted by the number of disease mutations within single-pass TMs (Table 1). Although many of these mutations are uncharacterized, the involvement of polar or small residues points toward changes in protein-protein interactions within the membrane.

Table 1.

Disease-Associated Single-Pass TM Mutations

Ectodysplasin H54T LLFLGFFGLSLALHLLTLCCYL X-linked hypohidrotic ectodermal dysplasia (Vincent et al., 2001)
Y
FXYD2 G41A LIFAGLAFIVGLLILLSRRF Primary hypomagnasemia
R (Meij et al., 2000)
TACI A181E VALVYSTLGLCLCAVLCCFLVAVA Common variable immunodeficiency
E (Castigli et al., 2005) (Salzer et al., 2005)
GPIX A146T GVLWDVALVAVAALGLALLAGLL Bernard-Soulier syndrome
T (Wang et al., 2004)
MPL W515L ISLVTALHLVLGLSAVLGLLLLRW Myelofibrosis with myeloid metaplasia
L (Pikman et al., 2006)
MPZ G134R YGVVLGAVIGGVLGVVLLLLLLF Charcot-Marie-Tooth disease
G138R R (Shy et al., 2004)
G138A R
A
Caveolin-3 P104L LLSTLLGVPLALLWGFLFACISF Hypertrophic cardiomyopathy
(mouse) L (Ohsawa et al., 2004)
Neu/ErbB-2 V664E VTFIIATVVGVLLFILVVVVGILI Oncogenic
(rat) E (Bargmann et al., 1986)
ErbB-2 I654V IISAVIGILLVVVLGVVFGILI Increased risk of breast cancer
(human) V (Frank et al., 2005)
FGFR1 Y372C YLEIIIYCTGAFLISCMVGSVIVY Osteoglophonic dysplasia
C379R C
R
FGFR2 S372C SPDYLEIAIYCIGVFLIACMVVTV Beare-Stevenson syndrome
Y375C C
C
FGFR3 G370C GSVYAGILSYGVGFFLFILVVAAV Thanatophoric dysplasia I, bladder cancer
S371C C Achondroplasia
Y373C C Achondroplasia
G375C C Hypochondroplasia
G380R C Multiple myeloma
V381E R Crouzon syndrome
G382D E Bladder cancer
A391E D
E
FGFR4 G388R IILYASGSLALAVLLLLAGLY Tumor progression
R (Li and Hristova, 2006)
TREM-2 K186N SILLLLACIFLIKILAASALWA Nasu-Hakola disease
N (Paloneva et al., 2002)
FcγRIIb I232T IIVAVVTGIAVAAIVAAVVALIY Lupus
T (Kono et al., 2005)
KIT F522C LFTPLLIGFVIVAGMMCIIVMILT Mastocytosis
C (Akin et al., 2004)
G-CSFR T617N IILGLFGLLLLLTCLCGTAWLCC Acute myeloid leukemia
N (Forbes et al., 2002)

Static Interactions

Activating Immune Receptors

The biological importance of static TM interactions is clearly illustrated by a system of modular receptors expressed on the surface of hematopoeitic cells. These complexes use a network of charged residues to connect at least one ligand-binding protein that generally lacks intracellular signaling domains with a second group of signaling adaptor proteins (Call et al., 2002; Feng et al., 2005, 2006). The best-characterized member of this family is the TCR. The ζζ-homodimer structure (Call et al., 2006) described above is just one component of the larger TCR complex. Positively charged Arg and Lys residues on the TCRαβ TMs interact with negatively charged Asp and Glu residues on the CD3δε and CD3γε heterodimers and the ζζ-homodimer (Call et al., 2002). These interactions are necessary for proper assembly of the complex in the ER and subsequent transport to the cell surface (Call et al., 2002).

This high degree of specificity is also present in the other activating immune receptors that interact with the signaling modules Fcγ, DAP10, and DAP12 (Table 2; Feng et al., 2005, 2006). The importance of these interactions is illustrated by a Lys to Asn mutation in the TREM-2 receptor that blocks assembly with the DAP12 adaptor protein and results in Nasu-Hakola disease (Paloneva et al., 2002). Like the TCR, the B cell receptor (BCR) also pairs a signaling dimer (Ig-αβ) with a ligand binding receptor (mIg). Although the Ig-αβ chains contain Glu and Gln residues that may be analogous to the Asp and Glu residues in the CD3 complexes, there are no corresponding positively charged residues in the mIg TM (Table 2). Recent studies (Dylke et al., 2007) point toward specific interactions between the mIgM and Ig-αβ TM domains and early mutagenesis to polar Ser, Thr, and Tyr residues support a role for polar interactions within the membrane (Grupp et al., 1993; Pleiman et al., 1994; Stevens et al., 1994).

Table 2.

TM Sequences from Ligand-Binding Immune Receptors and Their Interacting Signaling Modules

Ligand-binding module Signaling module
TCRα VIGFRILLLKVAGFNLLMTL ζ LCYLLDGILFIYGVILTALFL
VIGFRILLLKVAGFNLLMTL CD3ε VMSVATIVIVDICITGGLLLLVYYW
CD3δ PATVAGIIVTDVIATLLLALGVFCF
TCRβ TILYEILLGKATLYAVLVSALVL CD3ε VMSVATIVIVDICITGGLLLLVYYW
CD3γ ISGFLFAEIVSIFVLAVGVYFI
NKG2C VLGIICIVLMATVLKTIVLIPFL
KIR2DS2 (Type II TM, C-type lectin)
VLIGTSVVKIPFTILLFFLL
KIR3DS1 ILIGTSVVKIPFTILLFFLL
NKp44 LVPVFCGLLVAKSLVLSALLVWW
TREM-1 VILLAGGFLSKSLVFSVLFAVTL
TREM-2 LLLLACIFLIKILAASALWAAAW DAP12 VLAGIVMGDLVLTVLIALAVYFL
IREM-2 FLLVVLLKLPLLLSMLGAVFWV
SIRPβ1 LVALLLGPKLLLVVGVSAIYICW
SILRβ VALAVALKTVILGLLCLLLLWW
CD200RLa LIILYVKLSLFVVILVTTGFVFF
NKG2D PFFFCCFIAVAMGIRFIIMVAIW (Type II TM, C-type lectin) DAP10 LLAGLVAADAVASLLIVGAVFL
FcαRI LIRMAVAGLVLVALLAILV
NKp46 LLRMGLAFLVLVALVWFLV
ILT1 LIRMGVAGLVLVVLGILLF Fcγ LCYILDAILFLYGIVLTLLYC
GPVI LVRICLGAVILIILAGFLA
OSCAR LVRLGLAGLVLISLGALVTF
mIgM LWATASTFIVLFLLSLFYSTTVT
mIgG LWTTLSTFVALFILTLLYSGIVT Igα IITAEGIILLFCAVVPGTLLL
mIgA LWTTITIFITLFLLSVCYSATVT Igβ IILIQTLLIILFIIVPIFLLL
mIgD LWTGLCIFAALFLLSVSYSAALT

The mechanism of signal transduction by activating immune receptors may differ between family members and is beyond the scope of this review. Receptor clustering and conformational changes are likely important, but other modes have also been proposed and reviewed (Choudhuri and van der Merwe, 2007). Therefore, it is possible that these seemingly static associations between charged residues in the membrane, which are necessary for assembly, are flexible enough to propagate information across the bilayer.

Switchable Interactions

The TM helices described in this section participate with soluble domains in finely tuned equilibria that govern signal transduction across biological membranes. In each case, conformational changes are propagated through the membrane via changes in the oligomerization and/or orientation of the TM helices. Because they must be able to switch between states without large energetic penalties, it is not surprising that these proteins contain GASRight or GASLeft motifs that rely on van der Waals interactions, as opposed to strongly polar or charged residues. In fact, there are a number of activating disease mutations in Table 1 that place Cys, Asp, or Glu residues in the TM, which could lock the proteins in active conformations.

Ion channels represent one class of proteins in which conformational changes give rise to gating events that open and close the pore. Although work in this field is beyond the scope of this review, the M2 proton channel from influenza virus should be mentioned, because the transmembrane domain of this protein is a homotetramer formed by association of four copies of a single TM helix. Recent NMR and crystal structures have provided possible mechanisms for the pH-dependent conduction of the channel (Schnell and Chou, 2008; Stouffer et al., 2008).

Cytokine Receptors and Receptor Tyrosine Kinases

Cytokine receptors and receptor tyrosine kinases (RTKs) are unrelated families of proteins that regulate cell proliferation through evolutionarily convergent mechanisms involving ligand-dependent activation of intracellular kinases. The simplest model for their signaling is an equilibrium between inactive monomers and ligand-bound dimers. However, several bodies of evidence discussed below suggest that TM interactions may play important roles in more complicated equilibria (Figure 1).

In the case of the erythropoietin receptor (EpoR), which is a cytokine receptor that directly regulates the production of red blood cells, it has been hypothesized that TM interactions may be responsible for the apparent increased activity of the mouse receptor relative to the human receptor (Ebie and Fleming, 2007). The human receptor lacks an SaxxdLxxx heptad repeat that is found in the murine protein and is necessary for increased TM dimerization in vitro (Ebie and Fleming, 2007; Kubatzky et al., 2001; Ruan et al., 2004a) and for the ligand-independent oligomerization of inactive receptors and increased signal transduction at low ligand concentrations (Constantinescu et al., 2001b). This interaction is quite specific, and activation is believed to be coupled to rotation of the TM in the membrane (Constantinescu et al., 2001a; Seubert et al., 2003). In contrast to EpoR, the biological significance of TM interactions in normal RTK physiology remains unclear. However the prevalence of TM sequences consistent with GASRight packing and the number of activating disease mutations in growth factor receptor TMs (Table 1) are suggestive. These topics are discussed extensively in a recent review (Li and Hristova, 2006).

Integrins

Like activating immune receptors, such as the TCR, integrins are modular signaling complexes that recognize diverse ligands. There are 24 known integrin α/β heterodimers composed of combinations of 18 α subunits and eight β subunits (Bennett, 2005; Luo and Springer, 2006). Most integrins exist in equilibrium between an inactive resting state and an activated state capable of binding its extracellular ligands (Figure 4). Integrin TMs and their short cytosolic domains are in close contact in the inactive receptor, and activation is accompanied by their separation (Kim et al., 2003; Litvinov et al., 2004). This process is reversible and is only part of a larger network of linked equilibria involving integrin extracellular, transmembrane, and cytoplasmic domains and a host of interacting proteins. A push/pull mechanism (Li et al., 2005b) emphasizes these linked equilibria and postulates that any interaction or mutation that physically disrupts and αβ-chain TM helix-helix interactions will push the integrin toward the activated state, whereas cytoplasmic or TM proteins that preferentially interact with the cytoplasmic domains in either the inactive or active state of the integrin will pull the equilibrium toward one of these states. Most integrin TM domains contain GASRight-like sequences consistent with right-handed helical packing. In the case of the platelet integrin αIIbβ3, there are important TM interactions between a GX3G sequence in the α chain and bulky hydrophobic residues in the β3-chain (Li et al., 2005b; Luo et al., 2004, 2005; Partridge et al., 2004). Interestingly, the β3-chain contains an SX3A on the opposite face of the helix that may participate in as of yet unknown interactions.

Figure 4. Integrin TMs Make Switchable Heterodimeric Interactions.

Figure 4

(A) Integrins are held inactive by interactions between α- and β-chain TM and soluble domains. Interactions between cytosolic proteins and integrin cytosolic tails stabilize TM separation and activation of the extracellular integrin ligand-binding site (Bennett, 2005).

(B) A computational model of the β3-integrin TM domain packing against the GX3G motif of the αIIb-TM domain. Adapted from Li et al. (2005b).

TM-Regulating Peptides

Switchable interactions between TM domains can be regulated by other TM proteins that either inhibit or stabilize TM oligomerization. Viruses have evolved peptides that interact with the TM domains of growth factor and cytokine receptors to cause cellular transformation. The spleen focal forming virus (SFFV) protein gp55-P transforms cells through ligand-independent TM interactions with mouse EpoR (Constantinescu et al., 1999). The type 1 bovine papillomavirus (BPV) protein E5 also causes cellular transformation by activating the RTK platelet-derived growth factor β receptor (PDGFRβ) (DiMaio and Mattoon, 2001; Mattoon et al., 2001). E5 is a disulfide-linked type II TM protein that is predicted to make a homodimeric scaffold for the assembly of the PDGFβ receptor TMs into an antiparallel tetrameric coiled coil, bringing the receptor kinase domains in close proximity (Freeman-Cook et al., 2004, 2005).

Single-span TM proteins have also evolved to regulate ion channels through specific TM-TM interactions. Phospholamban (PLB) and sarcolipin (SLN) are homologous 52 aa and 31 aa TM proteins that regulate the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), which pumps calcium from the cytosol into the sarcoendoplasmic reticulum in cardiac muscle cells. Key residues within the TM domain of PLB drive pentamerization in detergents (Arkin et al., 1994). The solution NMR structure (Figure 5) of Oxenoid and Chou (2005) demonstrated a pentameric left handed-coiled coil with a heptad Leu/Ile zipper motif in which Ile residues occupy the “a” positions (Ile33, Ile40, and Ile47) and Leu residues occupy “d” positions (Leu37, Leu44, and Leu51). This motif had previously been shown to mediate tetramer formation in water-soluble coiled coils (Harbury et al., 1993) and had been proposed to stabilize the pentameric form of phospholamban B (Karim et al., 1998; Simmerman et al., 1996). Mutations to the TM that blocked oligomerization result in increased inhibition of SERCA by PLB (Fujii et al., 1989; Kimura et al., 1997), whereas mutations to the opposite face of the TM helix prevented inhibition of SERCA by PLB (Kimura et al., 1997), suggesting that SERCA inhibition may depend on the monomer form of PLB. Recent studies, however, are consistent with the PLB pentamer interacting with SERCA, suggesting that the mechanisms of transporter regulation may be more complicated than previously thought (Stokes et al., 2006). SLN also appears to make similar direct interactions with SERCA, though it is not known whether it too has a homo-oligomeric form, as does PLB (Hughes et al., 2007).

Figure 5. Phospholamban, Sarcolipin, and the FXYD Proteins Regulate Channel Proteins through TM Interactions.

Figure 5

(A) An NMR structure of the phospholamban homopentamer (Oxenoid and Chou, 2005). In magenta are residues that mediate interactions with the SERCA channel. They are on the opposite face from the Leu- and Ile-zippers that mediate pentamerization (red and orange, respectively).

(B) TM sequence of phospholamban showing important residues and the related TM sequence from sarcolipin.

(C) Alignment of the FXYD protein family TM sequences, which regulate the Na/K-ATPase. Highly conserved and functionally important residues are highlighted. A mutation in the second conserved glycine, Gly41Arg, in the FXYD2 TM results in dominant negative primary hypomagnesemia due to aggregation of FXYD2 in the cytoplasm and misrouting of the Na,K-ATPase (Meij et al., 2000).

The seven FXYD proteins (FXYD1–FXYD7) are evolutionarily unrelated to PLB and SLN but also function to regulate ion channels through TM interactions. They are tissue-specific modulators of the Na,K-ATPase, which governs the basic membrane potential that powers membrane transporters and maintains cell volume (Geering, 2006). With the exception of FXYD3, which has an uncleaved signal sequence, all FXYD proteins are single-pass type I TM proteins that contain an extracellular FXYD domain and conserved TM domains that are necessary for their functions (Figure 5C; Geering, 2006). Mutagenesis suggests that small residues at positions i, i+4, and i+11 along one face of the TM helix are important for interactions with the ATPase (Crambert et al., 2004; Li et al., 2005a) and that flanking residues specifically and differentially regulate Na+ and K+ affinity (Li et al., 2005a; Lindzen et al., 2003).

Exogenous Regulatory Peptides

A number of laboratories have used exogenous TM peptides to perturb signaling pathways believed to be dependent on TM interactions. Peptides containing the ErbB2 TM were shown to partially inhibit signaling at low concentrations of ligand in a GX3G-dependent fashion (Bennasroune et al., 2004). TM peptides that mimic the TCR TM have also been used to inhibit T cell function (Manolios et al., 1997; Quintana et al., 2007). Exogenous peptides based on the phospholamban (Afara et al., 2006) and FXYD2 (Zouzoulas et al., 2003) sequences also perturb SERCA and Na/K-ATPase function, respectively.

We have targeted the heterodimeric interface of the integrin αIIbβ3 with TM peptides from the wild-type integrin (Yin et al., 2006). Peptides that block the heteromeric association between αIIb and β3 should activate the integrin. Indeed, a peptide spanning the sequence of the αIIb TM specifically activates αIIbβ3, presumably by competing for this interface through homodimeric interactions. To design peptides with greater specificity and affinity, we developed a computational method for designing TM peptides to specifically target protein TM domains: Computed Helical Anti-Membrane Protein (CHAMP) (Yin et al., 2007). The GX3G-like motifs within the αIIb- and α_v_-integrin TM domains were targeted to demonstrate the specificity of the technique. Each of these integrin subunits is known to pair with a common β3 to create either αIIbβ3 or α_v_β3, which recognize different extracellular ligands. Sequence analysis showed that αIIb- and α_v_-integrin TM domains each have a high propensity to associate with a second helix in a parallel GASRight motif, indicating that a peptide that also had a strong propensity to adopt this motif would be likely to have a high affinity for the integrin TM sequence (Figure 2). Thus, backbones of helical pairs taken from the crystallographic database interacting with this geometry were chosen as potential templates for the design of a CHAMP peptide. The sequence of the integrin TM helical target was threaded onto one of the two helices, and the amino acid residues on the opposite helix was selected using a side chain repacking algorithm. The anti-αIIb and anti-α_v_ CHAMP peptides bound specifically to their intended integrin TMs and specifically activated receptor adhesion to their respective ligands.

Conclusions

As discussed above, there are a host of pathologic mutations in single-span transmembrane proteins that likely affect TM interactions. As the structural and sequence motifs that govern TM interactions become increasingly apparent, we will better understand the pathophysiology of these mutations. Similarly, we are beginning to better understand the roles of wild-type TM sequences in such medically important processes as hemostasis and cell proliferation. These structural and mechanistic insights are just now beginning to allow us to develop technologies such as CHAMPs and potentially even small molecules that can interact with transmembrane domains as molecular probes of biological function or new classes of targeted therapeutics.

Acknowledgments

We sincerely thank Joel S. Bennett, Jason Donald, and Alessandro Senes for their thoughtful discussions and helpful input during the preparation of this manuscript.

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