Marginally hydrophobic transmembrane α-helices shaping membrane protein folding.

REVIEWS Marginally hydrophobic transmembrane a-helices shaping membrane protein folding

Minttu T. De Marothy* and Arne Elofsson* Department of Biochemistry and Biophysics Science for Life Laboratory, Stockholm University, Solna, SE-171 21, Sweden Received 30 March 2015; Accepted 24 April 2015 DOI: 10.1002/pro.2698 Published online 13 May 2015 proteinscience.org

Abstract: Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. Keywords: membrane protein folding; hydrophobicity; marginally hydrophobic transmembrane helices; aquaporin 1

Introduction The majority of a-helical integral membrane proteins in both prokaryotic and eukaryotic cells depend on a

This review is adapted from portions of Minttu De Marothys PhD thesis. *Correspondence to: Minttu T. De Marothy, Department of Biochemistry and Biophysics Science for Life Laboratory, Stockholm University, Solna, SE-171 21, Sweden. E-mail: minttu. [email protected] or Arne Elofsson, Department of Biochemistry and Biophysics Science for Life Laboratory, Stockholm University, Solna SE-171 21, Sweden. E-mail: [email protected]

C 2015 The Protein Society Published by Wiley-Blackwell. V

translocon for membrane integration. For the past decade, structures of these protein-conducting channels have been solved and biochemical methods have characterized their function in membrane protein biogenesis. The sequence characteristics that define a membrane protein as well as dictates its topology have also been mostly deciphered. Membrane protein folding appears to follow a two-step process depicted in Figure 1(A). Individual transmembrane helices are recognized based on their hydrophobicity, and inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final

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Figure 1. Membrane protein insertion, topology and folding A) In the two-stage model, individual transmembrane segments are partitioned into the membrane one after an other in a sequential manner. B) In non-sequential membrane integration, some transmembrane segments are not initially recognized as such (depicted in red). As a consequence, some transmembrane helices enter the membrane in an opposite orientation and the protein adopts an intermediate topology.

conformation of the protein—a step we know much less about. Topologies established during membrane protein insertion are generally assumed to be relatively fixed. However, increasing number of solved membrane protein structures show far more variation in the hydrophobicities of transmembrane helices. Some transmembrane segments are not even initially recognized as such by the translocon. As a consequence, some membrane proteins have been reported to adopt intermediate topologies [see Fig. 1(B)]. Further, membrane proteins can undergo dramatic re-organizations, even inversion of transmembrane domains and/or translocation of extracellular loops. In one particularly interesting example, a single positively charged residue at the very C-terminus was sufficient to convert an established topology to an opposite one.1 We will begin this review by discussing the hydrophobic effect as it is paramount to understanding the formation of biological membranes, insertion of transmembrane segments and protein folding. Next, the composition of biological membranes is discussed as its role in membrane protein topology, folding and function has become increasingly important. The structure of the Sec-translocon and the roles of amino acids in membrane protein insertion are briefly discussed as both have been extensively reviewed before. Because the role of the lipid membrane is becoming more evident in influencing membrane protein structure and function, a significant portion of this review is devoted to describing biological membranes. Especially as the properties of amino acids found in transmembrane segments are dependent on their environment. The focus of this review is to describe marginally hydrophobic helices, mTMHs, that is, transmembrane a-helices that are

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not recognized by the translocon by themselves, and their role in shaping membrane protein folding.

The Hydrophobic Effect Water molecules are electric dipoles, where the electronegative oxygen has a partial negative charge and the small hydrogen atoms have a partial positive charge. As a consequence, they have dipole interactions with each other as well as to other molecules. The dipole interaction between a hydrogen atom bonded to an electronegative nucleus (nitrogen, oxygen, fluoride, and chloride in particular) and a second such nucleus is termed a hydrogen bond. The hydrogen bond is particularly strong, mainly due to the large dipole moment caused by the electronegative nuclei’s polarization of the X-H bond (where X is an electronegative atom and H a hydrogen), but also due to a partial covalent character.2 Liquid water forms a tightly hydrogen bonded network between the molecules (typically three hydrogenbonds/molecule3), and the relative strength of these interactions is demonstrated clearly by the textbook comparison (e.g., Pauling2) to hydrogen sulfide, which is gaseous at room temperature. Hydrogen bonds are the strongest intermolecular interaction between overall neutral molecules. A well-known observation “oil and water don’t mix,” is known as the hydrophobic effect in chemical terminology. If a non-polar molecule is placed in bulk water, it disrupts the hydrogen-bond network of the water, in effect creating a “bubble” around the non-polar molecule. The energetic cost of this may be accounted in two parts: First, the energy required by surface tension to create an interface and form a void in the water. Second, the energy gained from the weak van der Waals (vdW) interactions (Debye, London forces) that exist between the nonpolar

Marginally Hydrophobic Transmembrane a-Helices

molecules placed into the void and the surrounding water. Because attractive forces are much weaker than hydrogen bonds, the former term is guaranteed to dominate in the case of a purely non-polar molecule, and it will cost energy to place the non-polar molecule into the bulk water. The energetically favored situation is for the non-polar molecules to “lump together” into droplets or bubbles, and ultimately a separate phase, in order to minimize the interface area with the water. The nonpolar substance is immiscible in water. The term “hydrophobic” can be misleading. While bulky hydrophobic molecules may form strong intermolecular bonds (e.g., oils have a higher boiling point than water), they do not bond to each other more strongly than they do to water in terms of interfacial area.4 Nonpolar molecules interact with water through the Debye (induced dipole moment) and London (dispersion) interactions, while nonpolar molecules bind to each other through the weaker (but longer-range) London force alone. Therefore, the driving force behind the hydrophobic effect is not that non-polar molecules bind to each other more strongly than to water, but rather that water binds to itself more strongly than it does to non-polar molecules. Although a completely nonpolar molecule is immiscible, hydrophobicity is not an either or phenomenon. The more polar and hydrogen-bonding a molecule is in relation to its size, the more the energetic cost of creating a void in the water is offset. Hence methanol is entirely miscible in water,5 1-pentanol is weakly soluble, while 1-decanol is normally considered insoluble.

The hydrophobic effect and membrane lipids Lipid bilayers are composed of amphiphatic lipids, consisting of a hydrophilic head group and a hydrophobic acyl chain [see Fig. 3(A)]. Despite the head group, they are still largely immiscible in water, and therefore aggregate spontaneously. They will also arrange themselves with their head groups at the water interface, where those groups may form hydrogen and polar bonds to the surrounding bulk water. This lowers the interfacial energy sufficiently that a stable emulsion can be formed, rather than lipids aggregating into an “oily” phase. Surface tension will lead to the formation of a spherical miscelle, unless the acyl chains are sufficiently bulky for their steric repulsion to counter it. In the latter case a stable lipid bilayer membrane is formed, with oppositely oriented layers of the amphipilic lipids.

The hydrophobic effect and proteins Amino acids are polar compounds with good water solubility. Upon forming peptide bonds, the charged groups become the amide and keto groups of the peptide backbone, which are hydrophilic but substantially less so than the original charged groups.

De Marothy and Elofsson

The amino-acid side chains may be polar, charged or hydrophobic. In addition, the folding and geometry of the protein can make the various groups inaccessible from the surrounding medium. Therefore, proteins can exhibit wide range of hydrophobicity, both as a whole and in localized regions of the protein.

Biological Membranes Biological membranes are mainly composed of lipids and proteins. Together they form an essential barrier between living cells and their external milieu. In eukaryotes, they also allow compartmentalization of intracellular organelles. While membrane lipids have been seen as merely solvents for membrane proteins, the picture today is more complex; biological membranes are dynamic structures, that vary in their lipid, protein and carbohydrate composition coevolving and functioning together.6 Indeed, the role of lipids in membrane protein structure,7,8 topology,9 function,10 and in signaling11 have become evident.

Physical properties of lipids shape the characteristics of a biological membrane The lipid composition of membranes varies between different organisms, membranes, cell types and in time. Organisms can devote up to 5% of their genome towards lipid metabolism12 and the number of different lipids range from hundreds in bacteria to thousands in eukaryotes.13 While the combination of different lipid head groups shape the characteristics of the membrane surface, the physical properties of the membrane are largely dependent on both the head groups and fatty acid side chains.14 Cylindrical lipids are prone to forming bilayers and are abundant in biological membranes [Fig. 2(B)]. However, non-bilayer lipids are also present in the membrane; Cone-shaped lipids have head groups with a cross-sectional area smaller than their acyl chains [Fig. 2(C)]. They promote a negative curvature in membranes whereas lipids with a invertedcone shape tend to form micelles and promote positive curvature in membranes15 [Fig. 2(D)]. Ultimately, the ratio between bilayer- and nonbilayer forming lipids determines the intrinsic curvature of the membrane as well as shape and integrity.10,16 Additionally, it influences the membrane flexibility, which is important for fusion/fission events as well as for membrane protein function.6,17,18 Membrane fluidity depends on acyl chain composition. Saturated fatty acids have a linear acyl chain allowing it to pack tightly, whereas unsaturated fatty acids contain kinks resulting in increased fluidity. In addition, incorporation of rigid steroidic lipids, such as cholesterol, confer stability.16 Membrane fluidity is also dependent on temperature and

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Figure 2. Structure of a phospholipid. A) The structure of phospholipids is illustrate with phosphatidylcholine. The head group consists of the glycerol backbone (in green), linked through a phosphate (in cyan) to a polar or charged head group (in magenta). Here this head group is choline, but it can be replaced by for instance ethanolamine in phosphatidylethanolamine. The two remaining carbons of the glycerol are attached to two fatty acids through ester linkage. The fatty acids can be saturated or unsaturated. The saturation status as well as the properties of the head group determines the physiochemical properties of a phospholipid. B) Schematic representation of lipids with different overall shapes and on how the fatty acid structure influences membrane curvature.

some organisms can adjust their lipid composition in response to changes in temperature.19

The lipid environment in biological membrane is heterogenic The structure for a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer has been solved ˚using X-ray and neutron scattering data. The 30-A ˚ thick hydrophobic core is lined with a 15-A-thick interface region on each side. Transition from the core to the interfaces is not sharp and should be viewed as a zone with gradual change in hydrophobicity.20 This can also be envisioned in the favorability of different amino acid side-chains residing in a transmembrane helix.21 The interfacial regions vary between the two leaflets. While the lipids are uniformly distributed on both faces of the bilayer in the endoplasmic reticulum membrane and the Golgi apparatus, the two leaflets of plasma membranes are asymmetric.12 A common feature in many animal cells is that the extra cytoplasmic leaflet contains most of the phosphatidylcholine, sphingomyelin, and glycosphingolipids, while phosphatidylserine and phosphatidylethanolamine are enriched in the cytoplasmic

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leaflet.22 How the asymmetry is established and maintained is not well understood. In addition to the differences between leaflets, membranes can also exhibit lateral asymmetry. Membrane domains can be defined as short-range ordered structures (0.001–1.0 lm in diameter), enriched in particular lipids and proteins. This lateral heterogeneity has been related to characteristic functional properties23 and both protein- and lipid-based mechanisms for membrane domain formations have been described (reviewed in Ref. 24). For some time, lateral heterogeneity was assumed to be an eukaryotic feature but lipid domains were later also found in bacteria.25

The fluid mosaic model and beyond The fluid mosaic model describes how proteins and lipids are organized in biological membranes. It depicts the membrane as a two-dimensional viscous lipid matrix, within which freely diffusing membrane proteins are embedded in. The model assumes that membrane proteins are interacting with lipids mainly due to hydrophobic forces.26 However, more recent results have added new layers of complexity to this model. Specialized membrane domains and extramembraneous structures can limit motion and


Figure 3. Protein machinery involved in the membrane integration of a-helical membrane proteins. A) A cartoon over the SRPdependent cotranslational pathway. When the N-terminal signal sequence (orange) emerges, it is recognized by the Signal Recognition Particle (SRP, in green). The ribosome-nascent chain complex is brought to the translocon (cyan) through the interaction between SRP and SR-receptor (red). B) Bottom view of the translocon heterotrimer (pdb 1rh5). The alpha subunit (grey) forms a clam-shell like structure with the gamma subunit (purple) associated at its back. The beta subunit is shown in red. C) The loop between the 5th and 6th TMHs form a hinge, which allows the lateral opening of the channel. The plug (green) resides in the center of the channel, maintaining membrane permeability. The lateral gate formed by the 2nd (blue) and the 7th (gold) TMHs is highlighted.

lateral diffusion of membrane components and protein-lipid interactions can be essential for protein structure and function.24,27 Biological membranes are rich in protein— reported lipid:protein ratios vary depending on cell type but range between 18 and 80% protein by mass28—something that may influence the actual hydrophobicity of a biological membrane and consequently the flexibility of membrane proteins and the presence of hydrophilic amino acid residues in transmembrane segments.29 Along these thoughts, it has been suggested that the amino-acid sequence of transmembrane segments would reflect on the properties of the bilayers in which they reside.30 Lipid–protein interactions. So far the characteristics of a lipid bilayer have been discussed in relation to the physical and chemical properties of lipids. However, an important consideration is also how membrane proteins and lipids interact with each other. Several features of the membrane have been described to influence the function of membrane proteins: the thickness and phase of the lipid bilayer and the presence of a specific phospholipids.31 Hydrophobic mismatch. An ideal transmembrane domain would span the hydrophobic core of the membrane, which would require an a-helix with 20 amino acids. However, both longer and shorter


transmembrane segments exist and do not match the thickness of the lipid bilayer, a phenomenon termed hydrophobic mismatch. If the transmembrane helix is too long, hydrophobic side-chains are exposed to the aqueous environment. If the transmembrane helix is too short, hydrophilic amino acids reside within the membrane. Both situations are energetically unfavorable. To minimize the free energy of the system, long transmembrane helices may either tilt or distort through coils to compensate for the mismatch, thus preventing the exposure of hydrophobic side chains to the aqueous environment.32 It may also be possible for these hydrophobic regions to reside within a folded, extramembraneous part of the same protein, or protein–protein interactions may shield them from the aquaous environment. Another alternative is that the lipid tails around the protein extend by acyl chain ordering.33 When the transmembrane domain is shorter than the hydrophobic core of the bilayer, the tails of the surrounding lipids may compensate by chain disordering, resulting in compression.34–36 However, it remains unclear whether lipids in living cells can change the thickness of the membrane.37 Nevertheless, mismatch has been implicated in the functionality of several proteins27,38 and may also be reflected in membrane protein sorting in eukaryotes.30,39


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Membrane properties shaping protein function. Anionic phospholipids can regulate protein activity and structure40,41 as well as influence targeting to the membrane.42 Interaction with anionic phospholipids can be rather nonspecific; Unstructured regions enriched in arginine and lysine residues can form electrostatic interactions with a negatively charged lipid domain in the membrane.43 Rhodopsin function, in turn, can be related to the requirement for a relatively unstable membrane environment, brought about by lipids with small head groups and bulky acyl chains.44 The mixing of bilayer prone and non-bilayer lipids can cause the tail region to over-pack while the head group region remains under-packed. This curvature stress results in a higher lateral pressure in the middle of the membrane, which in turn may affect membrane protein structure. A transmembrane protein may relieve this stress by adopting an hourglass shape, a feature found in a large number of membrane protein structures.45 In addition, lipid composition of the membrane can direct the topology of at least some proteins in a nonspecific manner.14 Implications of specific lipid properties in membrane protein function. Some lipids interact specifically with a protein regulating its function. Because membrane proteins are generally solubilized in detergent solutions before crystallization, lipids are probably underrepresented in crystal structures. In addition, the lipid molecules present in the crystal may represent a few tightly bound lipid molecules and may therefore not represent typical protein–lipid interaction. Regardless, examples where the structure and function of a membrane protein is linked to a specific interaction do exist.31,43 For instance, the full activation of Protein Kinase C is dependent on a specific interaction between phosphatidylserine.46 Likewise, a conserved binding site for Phosphatidylinositol 4,5-bisphosphate (PIP2) has been identified in K1 channels and its functional importance was demonstrated in the Kv7.1 channel, where the coupling of the Kv7.1 channel voltage-sensing domain and the pore domain requires PIP2.40 A more general feature, such as the lipid head-group size, can also be important for protein function.10

Protein Machineries in the Biogenesis of a-Helical Membrane Proteins A common pathway for the transport, translocation, and insertion of a-helical membrane proteins is dependent on the Signal Recognition Particle (SRP).47 An overview of this pathway is shown in Figure 3(A). The major components are the cytosolic SRP, its membrane bound signal recognition particle receptor (SR)48 and the core of the secretase (Sec)

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translocon—all universally conserved in the three domains of life.47

The SRP-dependent pathway A N-terminal signal sequence is crucial for the transport of a nascent polypeptide chain to its target membrane. It is characterized by a hydrophobic core, surrounded by positively charged amino acids at its N-terminus and polar amino acids at its Cterminus. SRP is recruited to a ribosome-nascent chain complex (RNC) as soon as the signal sequence emerges from the ribosome exit tunnel,47 in eukaryotes, this leads to stalling of the ribosome. Formation of the RNC-SRP complex is followed by its association with the ER (eukaryotes) or plasma membrane (prokaryotes), mediated by the GTP dependent interaction between SRP and its receptor. Once at the membrane, the RNC complex is transferred on to the translocon.48 These events are presented schematically in Figure 3(A). The RNC and translocon are associated with each other such that the ribosomal exit tunnel is aligned with the translocon pore and the polypeptide synthesized by the ribosome can be fed into the translocon.49 The translocon possesses a dual nature, allowing both a passage across the membrane and into the membrane. Transmembrane segments are recognized and shunted sideways into the membrane bilayer, while polar sequences either remain in the cytoplasm or translocate to the periplasm/ER lumen. Because the translocon itself is passive, a driving force is required. In the cotranslational mode, this force is provided by GTP hydrolysis during polypeptide synthesis.50

The Sec translocon The Sec61abg-translocon is a heterotrimer and facilitates the translocation of secretory proteins across, and insertion of membrane proteins into the ER membrane [shown in Fig. 3(B,C)]. The functional homolog in bacteria is SecYEG and in archaea SecYEb, both which reside in the plasma membrane. Sec61a and c share high sequence similarity with SecY respectively SecE. Both crystal- and cryoelectron microscopy structures of SecY and Sec61 have revealed fascinating details about the channel structure and function.51–54 The model for translocon function in membrane protein biogenesis is based on both structural data as well as many biochemical studies.55,56 The a-subunit, a membrane protein with 10 transmembrane helices forms the protein-conducting channel. When viewed from the side, it has an hourglass-shape with a narrow pore composed of hydrophobic amino acids,51 through which secretory proteins and the loops of membrane proteins cross the membrane.57 From top view, Seca has a clamshell like shape with two halves formed by TMH1-5


and TMH6-10 [Fig. 3(B)]. The loop between TMH5 and 6 functions as a hinge, allowing the opposite side of the protein to open up towards the lipid bilayer51 [Fig. 3(C)]. This lateral gate is mainly formed by TMH2 on one side and by TMH7 on the other51 [Fig. 3(D)]. The opening allows exposure of a translocating polypeptide to the hydrophobic environment of the lipid bilayer, with the possibility of said polypeptide to partition into the membrane.52 The exact mechanism is unclear, but experimental and molecular dynamics simulation data suggests that the binding of a prospective transmembrane domain to the translocon could stabilize the open conformation of the lateral gate.58,59 The mammalian Secc and the prokaryotic SecE are single-spanning membrane proteins in most species, found associated to Sec61a on the back/hinge side in the crystal structure.51 The third, nonessential subunit (Sec61b/SecG) is usually a singlespanning protein in archaea and eukaryotes but has two transmembrane domains in bacteria.

Associated proteins The active translocon has been observed to be a multimer of Sec heterotrimers and associated with other proteins. However, the actual number of subunits and their identities have remained controversial.60,61 Cryo-EM reconstructions of native ribosometranslocon complexes suggested a complex with two dimers of the Sec61 heterotrimer and two tetrameric translocon-associated protein complexes (TRAP),60 while crosslinking studies indicate that a single Sec61 heterotrimer is responsible for protein translocation.61 Perhaps the controversy is in part due to versatility of the Sec-translocon, where the number and identify of interacting proteins is at least occasionally dependent on its substrate.62–64 The cotranslational insertion of many membrane proteins and bacteria seem to require YidC, a membrane protein with six transmembrane domains.65 In eukaryotes, the monomeric translocation-associated membrane protein (TRAM) may fulfill a similar function.66 Both TRAP and TRAM have been crosslinking to nascent peptide as they emerge from the translocon, possibly influencing integration into the bilayer66,67 or the topology.68 Two modifying proteins are also found in the close vicinity of the translocon, namely the signal peptidase complex69 and oligosaccharyl transferase.70

Properties of Amino Acids in Membrane Protein Structure Topology is used to describe a-helical membrane proteins. In this context it refers to the number and orientation of transmembrane helices as well as the location of the N- and C-termini of a protein. Membrane proteins tend to follow a predictable pattern of topological organization; anti-parallel transmem-


brane helices cross the membrane from one side to the other with hydrophilic loops alternating between cytoplasmic and non-cytoplasmic location. By now many properties directing the topology of a nascent polypeptide chain have been reported71,72; Transmembrane helices are recognized through sufficiently long and hydrophobic sequences of amino acids and the orientation is determined mainly by the distribution of positively charged amino acids according to the positive-inside rule.50

Hydrophobic effect and membrane proteinsdefining amino acid hydrophobicity Just as there is an energetic cost associated with introducing a non-polar molecule (or amino-acid side chain) into an aqueous environment, there is an energetic cost from introducing a polar molecule or side chain into the non-polar membrane interior, due to the loss of polar interactions or hydrogen bonding with water. The polar peptide back-bone opposes membrane insertion, even though the free energy cost can be reduced by secondary structure formation.73,74 Recent experiments with non-proteinogenic amino acids have demonstrated that the hydrophobic surface area of an amino-acid side chain is directly proportional to membrane insertion.75 In other words, the hydrophobic effect will promote partitioning of a peptide when said peptide contains a certain number of hydrophobic amino acid side-chains. In the cell, transmembrane helices are recognized by the translocon, based on the average hydrophobicity of a stretch of amino acids. Several experimental and computational studies of artificial systems, as well as in vitro, in vivo and statistical methods have been used to assess amino acid hydrophobicity.76 The biological hydrophobicity scale, also known as the Hessa scale, defines the individual contributions of amino-acid side chains in a position specific manner.21,77 Correlation between the different hydrophobicity scales is in general good. However, compared to other experimental scales, polar and charged side chains are tolerated unexpectedly well in the membrane.21,77 Concurrently, the hydrophobicity of proline varies significantly. It is hydrophobic in the GES and Wimley–White scale,76 but rather hydrophilic according to the Hessa scale. The later being understandable, given its helix-breaking nature.78 Overall, the differences between hydrophobicity scales may be attributed to the high abundance of proteins in biological membranes, compared to the uniformly hydrophobic environment composed of membrane mimics such as organic solvents.76

The properties of amino acids in membrane proteins To establish the Hessa scale, a series of artificial, potential transmembrane segments were designed


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and presented to the translocon in microsomal membranes. The in vitro expression system allowed a quantitative assessment of membrane insertion efficiency of the test segments.77 The probability of insertion conformed to a Boltzmann distribution, suggesting that the translocon-mediated insertion is an equilibrium process. Hence, the apparent free energy of insertion (DGapp ) can be calculated and used to express the potential for membrane insertion of a given polypeptide.50,77 By systematically varying the amino acid composition of these test segments, the molecular code for translocon mediated membrane insertion was deciphered.21,77,79 Similar studies were carried out in the E. coli inner-membrane,80 baby hamster kidney cells,77 and yeast.81 Hydrophobic and helix-forming amino acids promote membrane insertion. A transmembrane helix is exposed to a varying milieus in the lipid bilayer, also reflected in the statistical difference in amino acid distribution within it.45,82 However, which amino acids are present in transmembrane helices is not only determined by hydrophobicity and bulkiness but also in their abilities to form interactions with the protein itself and the environment they reside in. Isoleucine, leucine, phenylalanine, and valine promote membrane integration and dominate in the central region of a transmembrane helix. Cystein, methionine, and alanine are all at the threshold between those amino acids that promote membrane integration and those that do not.50,77 However, alanines are good a-helix-formers and therefore often found in transmembrane helices.82,83 Polar and charged residues are relatively rare in the membrane core, even though the increased number of crystal structures has resulted in many findings of charged residues and irregular secondary structure within the hydrophobic core,84 despite the high cost associated with placing polar residues having in the membrane.21,77 Charged amino-acid side chains in transmembrane helices. Long, aliphatic side chains allow charged amino acids to orient their side chains towards the interfacial region and to interact with lipid head groups.85,86 This so-called snorkeling may explain why polar groups are better tolerated in biological membranes than expected.87 In addition, intramembrane salt bridges have been found in some membrane proteins, with both structural and functional importance.88,89 Positively charged residues close to transmembrane helices are strong topogenic signals. In contrast, acidic residues are much less potent topology determinants and show no statistical preference for loops on either side of the membrane.83,90 However, they have been reported to influence topology under

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special conditions, such as when negative charges are present in high numbers,91 in close proximity of marginally hydrophobic transmembrane helices92 or when present within seven flanking residues from the end of a transmembrane helix.93 Aromatic amino acids are unequally distributed in transmembrane helices. Tryptophan and tyrosine are enriched near the ends of the helices, often referred to as the aromatic belt.45,83,94 They are believed to interact favorably with the lipid head groups and have been shown to anchor and stabilize tilt angles of transmembrane helices relative to the bilayer.95,96 Proline and glycine in transmembrane segments. Amino-acid sequences rich in prolines and glycines tend to form coils, that is, regions lacking regular secondary structure. The presence of coils allow higher degree of structural flexibility, creating swivels and hinges97 as well as reentrant regions.45,84,98 They are especially common in channels and transporters and are often required for function.98 Glycine is also involved in helix-helix interactions. The GxxxG motif is fundamental in helixhelix associations.99,100 Here, the two small glycine residues are separated by one turn creating a groove on the helix surface. This groove serves as a contact surface for another helix with the same motif. The GxxxG or variations of it (G/A/S)xxxGxxxG and GxxxGxxx(G/S/T) occur in more than 10% of all known membrane protein structures.99

Positive inside rule in establishing topology Studies on membrane proteins with known topology revealed a bias for positive charged residues in cytoplasmic loops. This uneven occurrence in amino acid distribution is generally referred to as “the positiveinside rule,” and has been shown to hold for most organisms.83,101,102 The presence of positive charges influence both the insertion and orientation of a transmembrane helix. For instance, a single arginine or lysine residue placed downstream of a transmembrane segment in Cin orientation can lower the apparent free energy of insertion by 0.5 kcal mol21. The effect is additive and dependent on the distance from the positively charge residue to the transmembrane segment.103 However, the effect may also contribute globally, as a single positive residue placed at the very C-terminus of the dual topology protein EmrE, could flip the topology of the entire protein.1 The exact mechanism behind the positive inside rule is not fully understood. Because the cellular conditions for membrane protein insertion vary within the three domains of life, the effect of positive charges may even differ between archaea, bacteria


and eukarya. Why the retaining effect of positive charges is more pronounced in E. coli than in microsomes104 has been explained with the electrochemical potential across the bacterial inner-membrane being stronger than that of the ER membrane. However, the positive-inside rule does apply both in the ER and in the bacterium Sulfolobus acidocaldarius, with a reversed membrane potential.105 Likewise, if the membrane potential was responsible, negatively charged residues would be expected to have a more pronounced topogenic effect. This does not seem to be the case.45,83,90 A perhaps more likely suggestion is that the anionic phospholipids prevent membrane passage of positive charges. Basically, the negative head-groups of anionic phospholipids form electrostatic interactions to positively charged residues in protein segments, retaining the loop in the cytoplasm.43,106,107 As anionic phospholipids are present in all membranes,43 this might explain the ubiquity of the positive inside rule. The ability to modify lipid content in model organisms,108 could offer a very interesting opportunity to test the influence of lipids regarding the positive-inside rule. Likewise, the translocon has been implicated in positive-inside rule; specific interactions with the translocon contribute to the orientation of the signal sequence.109,110

The two stage model and topology The knowledge of properties of transmembrane domains, topology signals and folding of a membrane protein are often combined into the two-stage model. In this model, topology is established during the first step, as helices are inserted individually into the membrane. The second stage consists of interactions between the membrane embedded helices, folding into the final tertiary structure.111,112 Although this model applies to many proteins, others show more complex behavior.84,113–115 The first stage-establishing topology. Topology is in general established during co-translational membrane integration.116 Hydrophobicity and the positive-inside rule appear to be the main determinants but other more subtle features may contribute as well.114,117–121 The second stage-membrane protein folding. Unlike soluble proteins, membrane folding of membrane proteins occurs in an environment that is different in its character (the aqueous extra- and intracellular environments and the hydrophobic core of the membrane).112 To add to that, the orientation of transmembrane domains is considered relatively fixed. One might expect the loops to be important in keeping the membrane protein together, but transmembrane helices are known to assemble into a functional protein without the presence of


loops.122,123 This suggests, that the native fold of membrane proteins is mostly stabilized by interactions between transmembrane domains. VdW—(specifically dispersion)—forces have been suggested to be major driving force for membrane protein folding.94,124 The amino-acid residues in transmembrane regions are in general more buried than residues in soluble proteins or extramembrane regions,112,125 resulting in higher numbers of vdW interactions even if not necessarily stronger ones. The backbones of transmembrane helices can also be involved in “nonconventional” aC-H.O hydrogen bonds, and have been suggested to contribute to helix-helix interactions.126,127 While their influence on stability remains controversial,128,129 they might be important for helices at close distances and be suitable for maintaining stability.130,131

Non-sequential Membrane Integration of a-Helical Membrane Proteins The text book version of a membrane protein is an a-helical bundle, where each of the hydrophobic transmembrane helices cross the membrane more or less parallel to each other and perpendicular to the membrane.132 The topology is assumed to form sequentially and to be relatively fixed once established [see Fig. 1(A)]. However, the increasing number of solved membrane protein structures show far more variation in the structural elements of membrane proteins.133 Transmembrane segments can vary significantly in length,45,86,117 adopt strongly tilted conformations,134 have kinks, polar groups and non-helical regions in the middle of the membrane135 as well as re-entrant regions—helices that span only a part of the membrane before looping back. Transmembrane segments are not necessarily recognized as such by the translocon, which can result in nonsequential membrane insertion. As a consequence, some proteins have been reported to initially insert in intermediate topologies [see Fig. 1(B)]. Also, the initially inserted transmembrane regions may differ from the membrane embedded regions in the final structure.84,134 In both of these cases, the final topology and functional structure is achieved through later realignment events. Concurrently, an increasing number of results suggest that topology may not be as fixed as previously thought. Membrane proteins can undergo dramatic reorganizations, including inversion of transmembrane domains and translocation of extracellular loops, in response to changed lipid composition in vitro and in vivo. Because this can occur without the involvement of a cellular machinery, the activation energy must be low for re-assembly.9,136 In relation to this, it is worth to note that some a-helical membrane proteins are able to spontaneously insert into


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the membrane, even though the membrane integration in vivo is generally aided by protein machineries.137,138

Marginally hydrophobic helices A marginally hydrophobic transmembrane helix (mTMH) can be defined as a transmembrane segment unable to insert into the membrane by itself. A surprisingly large fraction (>30%) of transmembrane helices in multi-spanning proteins of known three-dimensional structure have a high DGpred , suggesting that their insertion into the membrane would be unfavorable. They are therefore likely to depend on extrinsic sequence characteristics for membrane integration.21 While at least some of these helices can insert in the membrane surprisingly well, those with even higher DGpred above tend to require other parts of the same protein for efficient membrane integration.114,115,132,139–141 Even though polar residues in transmembrane regions hamper membrane integration, they are more conserved than other residues in transmembrane segments.135 This is in part explained by their functional involvement and their tendency of being buried within the structure.135,142 Interestingly, polar residues are more common in polytopic membrane proteins with many transmembrane domains. Membrane integration of mTMHs depend on sequence features extrinsic to the hydrophobic segment itself. Some features have been identified (illustrated in Fig. 5) and include charged aminoacid residues flanking the hydrophobic segment,21,103,114 interactions between polar residues in adjacent TMHs,143–145 neighboring helices with strong orientational preference146 and repositioning of TMHs relative to the membrane during folding and oligomerization.84 However, the mechanisms by which these events take place and how frequent they are is still unclear.

Consequences of mTMH in aqp1 folding

Figure 4. Topological rearrangements in AQP1 due to a mTMH. AQP1 is initially inserted into the membrane as a four-helix intermediate (top). TMH2 (in red) cannot be inserted into the membrane initially resulting in the wrong orientation of TMH3 (in blue). Further, TMH4 can now not insert into the membrane due to a strong positive-charge bias. For AQP1 to obtain its six transmembrane domain topology (bottom), the reorientation of TMH3 is required. This reorientation requires residue Arg93 (marked with red circle) to cross the membrane. The middle panel shows a proposed “R1-H3 shift”; according to this model, TMH3 can spontaneously shift out of the membrane core, initiating topological realignment and folding of AQP1.

Aquaporin 1 (AQP1) is part of the ubiquitous family of water channels with six transmembrane helices and two re-entrant loops with a central water conducting pore.147 When AQP1 topology was studied in Xenopus oocytes, it was shown to initially insert as a four-helix intermediate before folding into its final structure at a later stage148,149 (see Fig. 4). These results were regarded with some skepticism based on contradicting results from experiments in mammalian cells.150 However, this apparent controversy has been solved by the observation that the intermediate is less stable in mammalian cells.149 Biogenesis of the close homolog Aquaporin 4 (AQP4), occurs more conventionally, with sequential and co-translational insertion of each transmembrane segment. The sequence features underlying the different behavior of AQP1 have attracted a lot

of attention. In essence, the second transmembrane helix is not sufficiently hydrophobic to integrate into the membrane,151,152 which in turn results in altered behavior of the rest of the protein.152 For one, TMH3 will be inverted in its opposite orientation while TMH4 is unable to integrate into the membrane, due to a number of positively charged residues at its C-terminus. Transition from this intermediate to the final topology requires the 180 rotation of TMH3 as well as the translocation of the loops between helices 3-4 and 4-5 (Fig. 4). The entire protein has to be translated for this remarkable realignments to occur.149,151 These observations imply interesting and novel characteristics in membrane proteins: a dilemma between features

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Figure 5. Local sequence context can aid in the insertion of a mTMH (in red). A) Positive charges in cytoplasmic loops can contribute toward the free energy for membrane integration and promote membrane insertion of a mTMH. B) Orientational preference of a subsequent TMH (in blue), induced by for example positive charges, can pull a mTMH into the membrane. C) Specific interactions (stars) between a mTMH and another transmembrane helix can allow insertion of the helix-pair. D) Repositioning can allow a segment of lower hydrophobicity to enter the membrane, even though a more hydrophobic part is initially recognized by the translocon.

required for function on one hand, but simultaneously allowing the correct folding of the protein. AQP1 demonstrates clearly the consequences of a mTMH in a membrane protein. The residues rendering mTMH2 unable to integrate to the membrane are functionally important.152 At the same time, correct orientation is a prerequisite for adequate exposure of extramembranous domains on the functionally relevant side of the membrane. How does the cell solve this dilemma of simultaneously conserving functionally important residues but preventing misfolding? After all, not only is the production of a non-functional protein costly,153,154 it also poses a burden for cellular quality control along with protein degradation systems. Clearly there must exist a mechanism to ensure proper membrane integration—some sequence features and cellular machineries have indeed been implicated and are described below. AQP1 folding also proposes significant plasticity in membrane protein topogenesis and folding. Similar behavior has been observed for several polytopic membrane proteins.115,139,141,155 The ability to reorient transmembrane segments may not be restricted to membrane protein folding. E. coli SecG has been reported to undergo reversible topology inversion as part of its function.156 Even more so, at least three membrane proteins (LacY, PheP, and GabP) are known to undergo dramatic realignments upon changes in membrane lipid composition.9 The dramatic contrast between the topogenesis of AQP1 and AQP4 are dependent on surprisingly


few differences in their primary sequences. Two residues (Asn49 and Lys51) in AQP1 mTMH2 are responsible for its hydrophilic nature. When exchanged to the corresponding residues in AQP4 (a methionine and leucine), the transmembrane domains of AQP1 are inserted in a sequential manner.152 Another recently characterized difference lies in the nature of the region just before TMH3, which contains the helical section of a re-entrant region 1, the loop between the re-entrant region and TMH3, along with the N-terminal part of TMH3. In AQP4, this segment is highly hydrophilic while the corresponding region in AQP1 has the hydrophobicity typically associated with transmembrane segments. We were able to show how this difference may account for the flexibility in AQP1 topogenesis, since the third transmembrane helix of AQP1 may shift out of the membrane core simultaneously bringing in the preceding “R1-H3 loop” into the membrane.157

Cost of polar groups in the membrane The hydrocarbon core of biological membranes has been considered to be a “forbidden zone” for charged amino acids.158 However, the membrane structure derived from X-ray and neutron scattering data depicts a rather dynamic structure. Only the very center of the membrane is entirely hydrophobic, the rest is a mixture of polar lipid head-groups and water molecules. Consequently, the membrane may partially permit the presence and passage of polar groups.20


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In regards to mTMHs and the ability of extramembraneous regions to cross the membrane, it is of interest to learn how unfavorable hydrophilic groups in the membrane really are. Arginine is often thought of as the most hydrophilic of amino acids and it is frequent in transmembrane helices.158 Molecular dynamics estimations suggest a barrier around 17 kcal mol21 159 and experimental scales report values ranging between 12 and 1.8 kcal mol21.160 However, direct comparison of different hydrophobicity scales is not straight forward as they are often normalized in different ways.76 According to the Hessa scale, the cost of an arginine in the middle of an hydrophobic helix is only 2.5 kcal mol2177,161 and strongly position specific.161 Several mechanisms seem to decrease the free energy penalty for a charged group in the membrane. Charged residues with long side chains can snorkel toward the membrane interface and polar groups can draw lipid head-groups and water deep into the core.135,159,162 Additionally, arginine (and other charged residues) may partition into the membrane when the presence of a sufficient number of hydrophobic amino acids overcomes the cost.158

Sequence features implicated in the insertion of mTMHs Positive-inside rule. Flanking loops and neighboring helices can be important for the membrane integration of mTMHs.114 In accordance to the positive-inside rule, arginines and lysines in cytoplasmic loops contribute towards the apparent free energy of membrane insertion by 0.5 kcal mol21 of a transmembrane helix, see Figure 5(A).103 However, cytosolic loops with positively charged residues do not always improve insertion of a mTMH.114 Characteristics in one transmembrane helix can influence the insertion propensity of another. This phenomenon was first observed for the human band 3 protein, where the strong orientational preference of a transmembrane helix resulted in membrane integration of an upstream region.115 Previous studies have implicated both positive charges and hydrophobicity in the likelihood of a transmembrane helix adopting a particular topology.72,118,120,163 A recent, systematic study showed how orientational preference of a neighboring helix could both increase and decrease the insertion of a mTMH146 [see Fig. 5(B)]. What then, gives a transmembrane helix an orientational preference—apart from cytoplasmic positively charged amino-acids residues? Long and hydrophobic helices may favor Nout–Cin orientation, while short and less hydrophobic helices instead adopt an Nin–Cout topology.117–119 A hydrophobic transmembrane helix may spend a shorter time inside the translocon, thus not having enough time to reorient. A less hydrophobic transmembrane helix, on

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the other hand, may not move out from the translocon as fast allowing re-orientation. This “hydrophobicity signal” can, however, be overridden by positively charged residues at the N-terminus and vice versa.120,121 Specific interactions between polar groups. Specific interaction within or between helices can improve their propensity to insert into the membrane [see Fig. 5(C)]. As discussed before, polar or charge groups hamper insertion of transmembrane domains. However, helices with charged or polar residues can interact with each other before entering the lipid bilayer, effectively “hiding” the energetically unfavorable groups in inter-helical hydrogen bonds.144,145 Repositioning in the membrane. One turn in an ideal a-helix employs 3.6 amino acids per turn ˚ . To span with one turn having the height of 5.4 A the hydrophobic core of a lipid layer 20 residues are needed to form a sufficiently long helix. However, the length of transmembrane helices vary between 12 and 40 residues promoting reorganization of both proteins and lipids.45,86,117,163 While tilting of transmembrane domains can occur as a consequence of hydrophobic mismatch,32 it is also possibly that tilting is induced during membrane protein folding due to packing interactions. It may also enable polar and charged side chains to become transmembrane, allowing them to get buried in the protein rather than being exposed to lipids [see Fig. 5(D)].134,164 The glutamate transporter homologue from Pyrococcus horikoshii serves as an example; Its three-dimensional structure is complicated with both mTMHs and transmembrane helices disrupted by coils. In a previous study, the primary sequence was aligned with known secondary structures and a theoretical hydrophobicity profile. Overlapping regions with more hydrophobic character were identified and at least one those segments was shown to be more prone insert into the membrane.84 Similar behavior has been reported in the human Band 3 protein and the KAT1 voltage dependent K1-channel141,165 where polar interactions within the protein enables membrane integration of transmembrane segments.166

Can translocon influence hydrophobicity threshold or topology? The translocon has been suggested to have an impact on the insertion and topology of transmembrane helices. Conserved residues both in the translocon pore ring and the later gate do affect the insertion of signal sequences.167,168 Substitutionsensitive residues have also been identified in the lateral gate, where they could both increase and reduce the threshold hydrophobicity for transmembrane segment. However, this impact is strong only


for single-spanning membrane proteins and on the first transmembrane domain of a polytopic membrane protein.169 Additionally, an arginine to glutamate substitution at the plug domain of yeast translocon weakens the positive-inside rule.110 It will be interesting to see if similar results can be obtained for E. coli and mammalian translocons. Whether the translocon is big enough to allow a polypeptide to invert has remained controversial.51,60,170–172 Because transmembrane helices can stay in close proximity to the translocon116,173 it has been proposed that reorientation occurs adjacent to the translocon complex even if not in it.72 Then again, the high protein content60,61,69,70,174 alone might favor insertion and reorientation of membrane protein segments.7,116 The time point and location where transmembrane helices can form interactions with each other have also gained some attention. Helical hairpins might form as early as in the ribosome “folding vestibule”175 and might allow membrane insertion of a mTMH.144 Can the translocon accommodate more than one polypeptide chain? Experimental studies suggest it might145,176 In fact, many membrane proteins do linger within or close by the channel.116,177 The translocon associated proteins TRAM and TRAP have both been crosslinked to nascent polypeptides emerging from the translocon affecting both membrane insertion66,67 and topology68 of their substrates. The size of the translocon protein conducting channel is important considering the popular model for membrane protein insertion.50,51,77 While the cytoplasmic cavity of M. jannaschii SecY has a diameter ˚ , the narrowest part of the channel is only of 20–25 A ˚ 5–8 A wide. To accommodate an a-helix, the channel would have to expand.51 Interestingly, the central ˚ ) in the Thermotoga maripore appears wider (10 A tima SecY structure, where SecY interacts with SecA and SecG.172 The channel size has also been assessed experimentally. In one study, SecYEG was challenged with rigid molecules of varying size fused to known Sec-substrates. SecYEG allowed the unrestricted pas˚ ,171 which is larger sage of constructs up to 22–24 A than seen in crystal structures or estimated by molecular dynamics simulations.178 Given the conflicting results and the huge thermodynamic driving force for membrane protein insertion9,94,137 a rather interesting model has been proposed. Perhaps the transmembrane helices never fully enter the translocon channel but interact with the cytoplasmic membrane interface and slide into the membrane utilizing the translocon lateral gate.179 This would sit well with the observations of neighboring helices enabling the membrane integration of mTMHs.

The membrane environment The topology of a membrane protein can be remarkably dynamic and may be altered by changing its


lipid environment. When the zwitterionic membrane lipid PE is depleted from E. coli, the topology of LacY exhibits a partial and reversible topological inversion.136 This re-assembly is independent on any cellular machineries.9 While the behavior of LacY may be surprising, it should not be considered an anomaly. Lipid-dependent topological realignments have been shown to occur for other proteins as well9,106 and membrane lipids are important for both insertion and stability of many membrane proteins.17,18 Further, since most eukaryotic membrane proteins are initially inserted into the endoplasmic reticulum and subsequently targeted to the membranes of other organelles this might also be reflected in membrane protein folding. The lipid composition in the different membranes is reflected in the composition of transmembrane segments30 and while the the ER membrane is mainly composed of phosphatidylcholine, PE, and phosphatidylinositol, the plasma membrane is in turned enriched in phosphatidylcholine, sphingomyelin and contains cholesterol.12,180 Perhaps this feature is also reflected in membrane protein folding and topology.148–150

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