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ANRV343-BB37-02 ARI 24 April 2008 15:5 How Translocons Select Transmembrane Helices Stephen H. White1,2 and Gunnar von Heijne3,4 1 Department of Physiology and Biophysics and 2 Center for Biomembrane Systems, University of California, Irvine, California 92697-4560; email: [email protected] 3 Department of Biochemistry and Biophysics and 4 Center for Biomembrane Research, Stockholm University, SE-106 91 Stockholm, Sweden; email: [email protected] Annu. Rev. Biophys. 2008. 37:23–42 Key Words First published online as a Review in Advance on February 7, 2008 membrane proteins, membrane protein folding, membrane protein assembly, membrane protein stability, lipid-protein interactions, hydrophobicity scales The Annual Review of Biophysics is online at biophys.annualreviews.org This article’s doi: 10.1146/annurev.biophys.37.032807.125904 c 2008 by Annual Reviews. Copyright All rights reserved 1936-122X/08/0609-0023$20.00 Abstract Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine is the translocon, which works in concert with ribosomes to manage the orderly insertion of α-helical membrane proteins directly into the endoplasmic reticulum membrane of eukaryotes or into the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid membrane, where physicochemical interactions determine the final three-dimensional structure. Much progress has been made during the past several years toward understanding the physical chemistry of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider the connection between the physical principles of membrane protein stability and translocon selection of transmembrane helices. 23 ANRV343-BB37-02 ARI 24 April 2008 15:5 Contents INTRODUCTION . . . . . . . . . . . . . . . . . MEMBRANE PROTEIN STABILITY AND ASSEMBLY . . . Dynamic Nature of Fluid Lipid Bilayers . . . . . . . . . . . . . . . . . Membrane Protein Intrinsic Interactions . . . . . . . . . . . . . . . . . . . Membrane Protein Formative Interactions . . . . . . . . . . . . . . . . . . . STRUCTURE OF THE TRANSLOCON . . . . . . . . . . . . . . . . TRANSLOCON RECOGNITION OF TRANSMEMBRANE HELICES . . . . . . . . . . . . . . . . . . . . . . . A Biological Hydrophobicity Scale . . . . . . . . . . . . . . . . . . . . . . . . . . Position Dependence of Free Energies . . . . . . . . . . . . . . . Prediction of Transmembrane Helices . . . . . . . . . . . . . . . . . . . . . . . Membrane Insertion of Multi-Spanning Proteins . . . . . . Helix-Helix Interactions . . . . . . . . . . THE BIOLOGY-PHYSICS NEXUS . . . . . . . . . . . . . . . . . . . . . . . . . 24 24 24 26 27 29 30 30 32 32 33 33 36 INTRODUCTION MP: membrane protein Translocon: a heterotrimeric membrane protein found in all organisms that is directly responsible for inserting α-helical proteins into membranes TM: transmembrane MD: molecular dynamics 24 Prediction of the three-dimensional structure of α-helical membrane proteins (MPs) from amino acid sequences is a challenging and important problem. One key to solving this problem is to understand quantitatively the formative interactions in biological assembly, which is initiated by the concerted action of ribosomes and translocons. Another key is to understand quantitatively the intrinsic interactions of MPs with the lipid bilayer of the membrane. The formative and intrinsic interactions must share some common principles, because the ribosome-translocon machinery delivers MPs to bilayers in a manner that allows MPs to come quickly into equilibrium with the bilayer after assembly (44, 80). White · von Heijne We attempt in this review to describe what is presently known about these principles. We begin with an overview of the properties of lipid bilayers and the general principles of MP stability and assembly, with an emphasis on the latter. More extensive discussions of MP stability are available in several comprehensive reviews (47, 61, 67, 80). In two succeeding sections, we first review progress toward understanding the structure and function of the translocon machinery, and then review progress toward understanding the code the machinery uses to recognize transmembrane (TM) segments of MPs. Finally, we discuss the inherent physicochemical connection between the formative and intrinsic interactions. MEMBRANE PROTEIN STABILITY AND ASSEMBLY Dynamic Nature of Fluid Lipid Bilayers The lipid bilayers of cell membranes must be in a fluid state for normal cell function. The characteristically high thermal disorder of this state precludes direct three-dimensional determinations of bilayer structure. But the onedimensional crystallinity of multilamellar bilayers dispersed in water or deposited on surfaces permits the distribution of matter along the bilayer normal to be determined by diffraction methods. For example, the combined use of X-ray and neutron diffraction measurements (76, 77) results in a structure consisting of a collection of time-averaged TM probability distribution curves of water and lipid component groups (e.g., carbonyls and phosphates), representing projections of their three-dimensional thermal motions onto the bilayer normal. Figure 1a shows the liquid-crystallographic structure of an Lα -phase dioleoylphosphatidylcholine (DOPC) bilayer (81) and Figure 1b is a snapshot from a molecular dynamics (MD) simulation of the same system. Three features of this fluid bilayer structure are important. First, the widths of the ARI 24 April 2008 15:5 probability densities reveal the great thermal motion. Second, the combined thermal thicknesses of the interfaces (defined by the distribution of the waters of hydration) are approximately equal to the 30 Å thickness of the hydrocarbon core (HC) of the bilayer. The thermal thickness of a single interface (∼15 Å) can easily accommodate an α-helix parallel to the membrane plane. The common lollipop cartoons of bilayers that assign diminutive thicknesses to the interfaces are thus misleading. Third, the interfaces are highly heterogeneous chemically. A polypeptide chain in an interface must experience dramatic variations in environmental polarity over a short distance because of the steep changes in chemical composition (79) (Figure 1c). MD simulations of bilayers, which provide dynamic three-dimensional information at the atomic level, are rapidly becoming an essential structural tool for examining lipidprotein interactions at atomic scales (3, 22, 57, 59, 68). The future offers the prospect of combining one-dimensional bilayer diffrac- tion data with MD simulations in order to arrive at experimentally validated dynamic, three-dimensional structures of fluid lipid bilayers (Figure 1b) with embedded proteins and peptides (6, 7). A movie constructed from an MD simulation of a fluid DOPC a Interface Hydrocarbon core HC: hydrocarbon core Interface Bulk water Probability ANRV343-BB37-02 Waters of hydration -40 0 -20 Methyls Methylenes Double-bonds l Carbonyls arbo Glycerol l cer Phosphate hosp Choline h lin 40 20 Distance from bilayer center (Å) b −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 1 c 0.05 Charge density The liquid-crystallographic structure of a fluid dioleoylphosphatidylcholine (DOPC) bilayer. (a) The structure of a fluid DOPC lipid bilayer (81) consists of a collection of transbilayer Gaussian probability distribution functions representing the lipid component groups of the bilayer unit cell. The areas under the curves correspond to the number of constituent groups per lipid represented by the distributions (e.g., one phosphate, two carbonyls, four methyls). (b) Molecular graphics image of DOPC taken from a molecular dynamics simulation by Ryan Benz. The color scheme is given in panel a. The image was prepared by S. White using Visual Molecular Dynamics (36). Two of the lipids are shown in space-filling representation. (c) Polarity profile ( yellow curve) of the DOPC bilayer (above) computed from the absolute values of atomic partial charges (79). The end-on view in panel c of an α-helix with diameter ∼10 Å, which is typical for membrane protein helices (9), shows the approximate location of the helical axis of the amphipathic helix peptide melittin (35). The figures in panels a and c have been adapted from reviews by White & Wimley (78–80). 0.04 0.03 α-helix 0.02 0.01 0.00 -40 -20 0 20 40 Distance from bilayer center (Å) www.annualreviews.org • Translocons and Transmembrane Helices 25 ANRV343-BB37-02 ARI 24 April 2008 15:5 bilayer can be viewed at http://blanco. biomol.uci.edu/Bilayer Struc.html. Membrane Protein Intrinsic Interactions Because MPs are equilibrium structures, their intrinsic interactions can be described by any a convenient set of experimentally accessible thermodynamic pathways, irrespective of the biological synthetic pathway. One particularly useful set of pathways is the so-called fourstep model (80) (Figure 2a), which is a logical combination of the early three-step scheme of Jacobs & White (37) and the two-stage model of Popot & Engelman (60). Although these b 4 Whole-residue interfacial scale Partitioning Folding Insertion ΔGaaIF (kcal mol-1) Interface HC core Association Coupled c ΔGaaWW (kcal mol-1) 2 1 0 I L F V C M AW T Y G S N H P Q R E K D -1 Peptide bond -2 d 4 3 3 Wimley-White whole-residue n-octanol scale TM α-helix Side chains Backbone ΔG = -10 ΔGsc = -36 ΔGbb = +26 2 1 0 I L F V C M AW T Y G S N H P Q R E K D -1 Peptide bond -2 (kcal mol-1) Figure 2 Energetics of peptide interactions with lipid bilayers. (a) Schematic representation of the four-step thermodynamic cycle of White & Wimley (80) used to examine the energetics of membrane protein stability through studies of small, water-soluble peptides (43, 85–87). The association of transmembrane (TM) helices is probably driven by van der Waals interactions, giving rise to knobs-into-holes packing (23, 47, 48, 63). (b) The Wimley-White (WW) interfacial hydrophobicity scale determined from measurements of the partitioning of short peptides into phosphatidylcholine vesicles (86). (c) The WW octanol hydrophobicity scale determined from the partitioning of short peptides into n-octanol (83). The free-energy values along the abscissa in panels b and c are ordered in the same manner as in Figure 4e. (d ) The energetics of TM helix insertion (38, 87) of glycophorin A estimated from the free-energy contributions of the side chains (Gsc ) and backbone (Gbb ). The net side chain contribution (relative to glycine) was computed using the n-octanol hydrophobicity scale (83). The per-residue cost of partitioning a polyglycine α-helix is +1.15 kcal mol−1 (38). Figures adapted from reviews by White and colleagues (72, 74). 26 White · von Heijne ANRV343-BB37-02 ARI 24 April 2008 15:5 pathways do not necessarily mirror the actual biological assembly process of MPs, they are nonetheless useful for guiding biological experiments, because they provide a thermodynamic context for biological processes. The objective of describing the stability of MPs by means of any of these schemes is to determine experimentally the thermodynamic constraints on MP structure formation. Each of the steps in the four-step model has been intensively studied by several laboratories during the past 15 years (1, 14, 47, 61, 67, 74, 80). Here we focus primarily on the energetics of helix stability as established from measurements of water-to-bilayer and water-to-octanol partitioning free energies of model peptides, summarized in Figure 2b,c (83, 84, 86). The most fundamental conclusion from these studies is that the unfavorable thermodynamic cost (GCONH ) of partitioning peptide bonds into membranes can be dramatically reduced by the formation of secondary structures (43, 85), because the partitioning free-energy GHbond of hydrogen-bonded peptide bonds is considerably lower than GCONH . For example, computational studies (4, 5) of peptides in bulk alkanes suggest that GCONH for water-toalkane transfer is +6.4 kcal mol−1 , compared to only +2.1 kcal mol−1 for GHbond . The per-residue free-energy cost of disrupting hydrogen bonds in an alkane is therefore about 4 kcal mol−1 , meaning that a 20-amino-acid TM helix would cost 80 kcal mol−1 to unfold within the membrane hydrocarbon (ignoring the interfacial gradients). This explains why unfolded polypeptide chains cannot exist in a TM configuration. As discussed in detail elsewhere (38, 74, 80), GHbond sets the threshold for TM helix stability (Figure 2d ). The free energy of transfer of nonpolar side chains dramatically favors helix insertion, whereas the transfer cost of the helical backbone dramatically disfavors insertion. What is the most likely estimate of GHbond ? The practical number is the cost Ghelix glycyl transferring a single glycyl unit of a polyglycine α-helix into the bilayer HC. The best estimate is +1.15 kcal mol−1 (38), which fortuitously corresponds to the cost of transferring a random-coil glycyl unit into noctanol (83). This finding led to a systematic evaluation of the Wimley-White (WW) noctanol as a hydrophobicity scale for predicting TM helices (38). The prediction accuracy exceeded 95%. Membrane Protein Formative Interactions Proteins destined for TM export (translocation) or insertion are generally managed by the concerted action of translating ribosomes in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of eukaryotes or in the plasma membrane of bacteria. The operating principles for the machinery of MP assembly (8, 15, 19, 40, 58, 75) are summarized in Figure 3a. The critical MP component of the translocon complex is heterotrimeric Sec61 in eukaryotes or the highly homologous SecYEG in bacteria. Cryo-EM image reconstructions (Figure 3b) of native ribosome-translocon complexes (52) suggest that the complex is likely composed of two dimers of the Sec61 heterotrimer and two copies of the tetrameric translocon-associated protein (TRAP). At least three other proteins associate closely with the translocon complex but do not seem to be part of the ribosometranslocon complex seen in the image reconstructions. These are the translocating chainassociated membrane protein (TRAM) (16, 25); the signal peptidase complex (SPC) (21), which cleaves signal sequences; and oligosaccharyl transferase (OST) (11), which Nglycosylates -Asn-X-Ser/Thr-sites on membrane and secreted proteins. The translocon complex acts as a switching station: Secretory proteins are allowed to pass straight through into the ER lumen or the bacterial periplasm (secretion), whereas TM segments of MPs are shunted sideways into the membrane bilayer. Deciphering the code that the translocon uses for selecting elongating segments for TM insertion is of www.annualreviews.org • Translocons and Transmembrane Helices WW: Wimley-White Translocon complex: an assembly of membrane proteins, including one or more translocons, upon which the ribosome docks SecYEG: translocon in the plasma membrane of eubacteria. Y, E, and G correspond, respectively, to the α, γ, and β subunits of Sec61 Sec61 or Sec61αβγ: translocon in the endoplasmic reticulum of eukaryotes 27 ANRV343-BB37-02 ARI 24 April 2008 a 15:5 Ribosome 1 b SRP mRNA Rib Ribosome Alu dom domain Exit tunnel RNA Polypeptide S S dom domain Emerging signal L 2 Membrane Elongation arrest Translocon complex 3 Docking Cytoplasm SR Translocon Exoplasm 4 Signal transfer and elongation N C Translocon N C N C c d Hydrophobic Hyd drophobic collar co colla ar Ribosom Ribosome (bac (back) Secβ Sec cβ β SecY Se ecY TM7 T M7 M TM7 7 TM2 TM TM2B 2B SecE SecE E Se E Exit 28 White · von Heijne (fro (front) ont) TM TM2B M2B T TM2 TM2A 2A Plu Plug ug ANRV343-BB37-02 ARI 24 April 2008 15:5 fundamental importance for understanding the folding of MPs (see below). But the selection of TM segments is only the first step in the complex process of gathering the TM segments together to form the native protein structure (12, 65, 66). Comparisons of the SecYEβ crystallographic structure with cryo-EM reconstructions (52) suggested that the heterotrimers form a tetramer arranged as a dimer-ofdimers ordered in a back-to-back configuration (“back” is defined in Figure 3c). No nascent peptide was observed in the crystallographic structure, which is thus assumed to be STRUCTURE OF THE in a closed state. Disulfide cross-linking experTRANSLOCON iments (10), however, revealed that elongating The key protein of the eukaryotic translo- chains pass through the so-called hydrophocon complex—the one that acts as the switch- bic collar in the middle of SecY (Figure 3d ), ing station—is heterotrimeric Sec61αβγ suggesting that translocon-mediated protein (SecYEG in eubacteria, SecYEβ in archaea) export and membrane insertion involve at any (56). The Sec61 α-subunit has 10 TM he- particular time only one of the SecY/Sec61 lices, whereas β and γ typically have 1 TM heterotrimers in the translocon complex. The helix (eubacterial SecE has 3 helices and SecG broad purpose of the posited tetrameric assohas 2 TM helices). Van den Berg et al. (70) ciation of SecY/Sec61 may be to provide an have determined the crystallographic struc- assembly platform that enables the ribosome ture of SecYEβ from Methanococcus jannaschii and other members of the Sec family to seat a resolution of 3.8 Å. It is shown embed- crete or insert nascent chains (55). Figure 3c shows SecYEβ from the viewded in a lipid bilayer in Figure 3c,d. The images are snapshots from an MD simulation point of the ribosome and Figure 3d shows of the heterotrimer embedded in a palmitoy- a view parallel to the membrane. The 10 TM helices of SecY are arranged to form an loleoylphosphatidylcholine bilayer (73). ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− SecYEβ: translocon in the plasma membrane of archaea Figure 3 Membrane protein assembly. (a) The machinery of membrane protein assembly. (Step 1) A ribosome translating the mRNA of a protein targeted for secretion across or insertion into membranes and a signal of a recognition particle (SRP), which is a GTPase. The structures of ribosomes are reviewed in References 18 and 62, and the structure of SRP is reviewed in Reference 54. (Step 2) The ribosome and SRP recognize a signal peptide as it emerges from the ribosome exit tunnel, bind together, and cause arrest of elongation in eukaryotes (27, 28, 82). (Step 3) The ribosome-SRP complex binds to the membrane-bound SRP receptor (SR), another GTPase that associates dynamically with the translocon. Prokaryotes use a simplified SRP (Ffh) and SR (FtsY) that associate to form a quasi-twofold symmetrical dimer (20). The binding of SRP to SR causes reciprocal stimulation of their GTPase activities, causing transfer of the signal peptide to the translocon and resumption of elongation (2). (Step 4) Proteins targeted for translocation are secreted into the periplasm (bacteria) or endoplasmic reticulum lumen (eukaryotes), whereas the stop-transfer signals of MPs are transferred to the membrane bilayer. (b) Cryo-EM image of the canine ribosome-translocon (Sec61) complex. The small and large ribosome subunits are indicated by S and L, respectively. Modified with permission from figure 4 of Reference 52. (c) Structure of a single SecYEβ closed-state translocon heterotrimer from Methanococcus jannaschii (70) that has been embedded in a palmitoyloleoylphosphatidylcholine lipid bilayer (red, headgroups; white, acyl chains) using molecular dynamics (MD) methods. SecY is viewed from the ribosome along the bilayer normal. The front and back of the protein are indicated. Sec Y is composed of 10 transmembrane (TM) helices. Helices 1-5 are colored dark purple, except for TM2B, which is red. Helices 6–10 are colored orange except for TM7, which is colored red. The presumed lateral exit from the TM7/TM2B lateral gate is indicated. (d ) SecYEβ viewed along the bilayer plane toward the lateral gate through which nascent TM helices are believed to move into the bilayer. The TM2A plug helix apparently seals the translocon in the absence of nascent peptide. The images in panels c and d were prepared from an MD simulation, courtesy of A. Freites. Both molecular graphics images were produced using Visual Molecular Dynamics (36). www.annualreviews.org • Translocons and Transmembrane Helices 29 ANRV343-BB37-02 ARI 24 April 2008 15:5 inverted “U” (Figure 3c), with TM helices 1– 5 forming one leg and helices 6–10 forming the other leg. The two sets of helices have a pseudosymmetric twofold rotation axis in the plane of the membrane and are connected at the back by an external loop. This loop and the single TM helix of SecE prevent lipids from contacting the interior of SecY from the backside. The only possible opening from the interior into the lipid bilayer is through the so-called lateral gate formed by TM2B and TM7 (Figure 3c,d ), which is hypothesized to control passage of nascent TM helices into the bilayer from the hourglass-shaped waterfilled interior of SecY (Figure 3d ). The two halves of the hourglass are separated by a ring of hydrophobic residues (hydrophobic collar) that are believed to act as a seal around the elongating chain. Sitting just below the hydrophobic collar is a short helix (TM2A) that apparently acts as a plug to block passage of small molecules through the translocon in the closed state. Van den Berg et al. (70) hypothesized that the plug is displaced by nascent protein translocation. But the necessity for the TM2A plug for sealing the hourglass in the absence of a translocating nascent chain was discounted in a study of a so-called plugless Sec61/SecY mutant (41, 49), because excision of TM2A had no effect on the viability of yeast cells. Remarkably, however, a crystallographic study of plugless SecY (45) showed that SecY restructures itself in the absence of TM2A to form a new plug! The image of SecYEβ in a lipid bilayer (Figure 3c,d ) is entirely consistent with the idea that TM helices move into the lipid membrane from the water-filled, proteinconducting channel by a simple partitioning process, as suggested by cross-linking studies of nascent chains (29, 50). In such a scheme, sufficiently hydrophobic helices prefer the bilayer, whereas more-polar helices favor the translocon and ultimately the aqueous phase. That is, the translocon and the lipid bilayer work in concert to decipher the code for TM helices embedded in the amino acid sequence. If this view is correct, then the big question concerns the code for deciphering the process. Answers to this question should lead to major improvements in the prediction of the membrane protein structure. TRANSLOCON RECOGNITION OF TRANSMEMBRANE HELICES A Biological Hydrophobicity Scale Insights into the process of TM helix insertion have been obtained by Hessa et al. (32), who used an in vitro expression system (64) that permits quantitative assessment of the membrane insertion efficiency of model TM segments (Figure 4). Specifically, −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 4 Integration of designed transmembrane (TM) segments (H-segments) into the endoplasmic reticulum (ER) using dog pancreas microsomal membranes. This system was used to explore systematically the hydrophobicity requirements for TM helix integration via the Sec61 translocon (32). (a) Wild-type leader peptidase (Lep) from E. coli has two N-terminal TM segments (TM1, TM2) and a large lumenal domain (P2). H-segments, flanked by glycosylation sites (G1, G2), were inserted between residues 226 and 253 in the P2 domain. For H-segments that integrate into the membrane, only the G1 site is glycosylated (left), whereas both the G1 and G2 sites are glycosylated for H-segments that do not integrate into the membrane (right). Redrawn from Reference 32. (b) An example of sodium dodecyl sulfate gels used in the in vitro determination of the extent of glycosylation of Lep/H-segment constructs in the absence (−RM) and presence (+RM) of dog pancreas rough microsomes. (c) Equations used by Hessa et al. (32) for the analysis of gels of the type shown in panel b. (d ) Mean probability of insertion, p, for H-segments with n = 0 −7 Leu residues. The curve is the best-fit Boltzmann distribution, which suggests equilibrium between the inserted and translocated states of the H-segments. (e) Biological Gaa app scale derived by Hessa et al. (32) from H-segments, with the indicated amino acid placed in the middle of the 19-residue hydrophobic stretch. ( f ) Correlation between Gaa app and the WW octanol ) (Figure 2c). Data in panels b–e are replotted from Reference 32. free-energy scale (Gaa WW 30 White · von Heijne ARI 24 April 2008 15:5 (G1 and G2) (Figure 4a) can occur only in the lumen of the RMs, H-segment TM insertion could be distinguished from secretion by simple gel assays (Figure 4b). The relative fractions of singly (1g) and doubly (2g) glycosylated molecules allow quantitative assessment of insertion versus secretion they examined the integration into membranes of dog pancreas rough microsomes (RMs) of designed polypeptide segments (Hsegments) engineered into the lumenal P2 domain of the integral MP leader peptidase (Lep) (Figure 4a). Because glycosylation of the engineered Asn-X-Ser glycosylation sites a b P2 G2 Translocated G1 66 kDa H G1 46 kDa G1 + G2: translocated, 2g G1 only: inserted, 1g Background Inserted ER Lumen H TM2 TM1 30 kDa Cytoplasm P1 G2 MW -RM +RM standards P1 P2 c d 1.0 Probability of insertion: Probability (p) P = f1g/(f1g + f2g ) Apparent equilibrium constant: Kapp = f1g /f2g Apparent free energy of insertion: ∆Gaaapp = -RTlnKapp 0.8 0.6 0.4 0.2 H: GGPG-LnA19-n-GPGG 0 0 1 2 3 4 5 6 7 8 Number of leucine residues (n) e f 4 ΔGaaapp (kcal mol-1) 4 3 E GGPG Xaa GGPG 2 1 0 -1 I L F V CM AWT Y G S N H P Q R E K D Amino acid ΔGaaww (kcal mol-1) ANRV343-BB37-02 3 H 2 R G 1 A 0 C V -1 -2 -3 -1 D K I L N Q T S M P Y F W 0 1 2 3 ΔGaaapp (kcal mol-1) www.annualreviews.org • Translocons and Transmembrane Helices 31 4 ANRV343-BB37-02 ARI 24 April 2008 Apparent free energy (Gapp ): an operational term for quantitating the favorability of transferring a polypeptide segment from the translocon into the membrane 15:5 (Figure 4c). The first experiments, carried out using H-segments of the form GGPG(Ln A19−n )-GPGG with n = 0 to 7, revealed that the probability of insertion, p(n), conformed accurately to a Boltzmann distribution (Figure 4d ). This showed that transloconmediated insertion has the appearance of an equilibrium process. Given this key observation, the insertion of H-segments was quantitated using the apparent free energy of insertion (Gapp ) (Figure 4c ). A biological hydrophobicity scale (Gaa app ) (Figure 4e) could be derived from studies in which each of the 20 naturally occurring amino acids were placed in the middle position of H-segments containing various numbers of Leu and Ala residues chosen to maintain p ≈ 0.5 (Gapp ≈ 0), which is the region of maximum sensitivity of the assay (Figure 4e). Considering the complexity of the biological system, the scale correlated surprisingly well (Figure 4f ) with the WW octanol scale (Figure 2c). Their overall high correspondence implies that the recognition of TM segments by the translocon likely involves direct interaction between the segment and the surrounding lipid (29), which seems reasonable in light of Figure 3d. Position Dependence of Free Energies Does Gaa app vary with position within the Hsegment? To answer this question, Hessa et al. (32) performed position scans of two types: single- and pair-scans. In the simpler singlescan, an amino acid of interest was placed at different positions in the H-segment sequence and Gapp was determined. The dramatic results from an Arg scan are shown in Figure 5a (34). Similar results were found for Lys, Asp, and Glu scans. The strong dependence on position must be related to the relative ease of snorkeling of the charge group to the wet bilayer interface (46)—the farther the charge is from the interface, the greater the energetic cost. The strong position dependence of Arg explains why it is possible for Sec61 32 White · von Heijne to insert the KvAP S4 voltage-sensing helix, which contains four Arg residues, across the ER membrane with Gapp ≈ 0 (34). An MD simulation of S4 across a lipid bilayer (24) showed that the arginines snorkel to the bilayer interface to form salt-bridges with the phospholipid phosphates and hydrogen-bond networks with water (Figure 5b). In pair-scans, a pair of residues of a given kind was moved symmetrically from the center of the H-segment toward its N and C termini to preclude the possibility of a shift in helix position across the membrane. Pairscans of charged residues were consistent with single-scans, suggesting that helix shifts were not significant. Pair-scans of the aromatic residues, which have preferential interactions with the bilayer interface (42, 86, 88), gave another insight into TM helix insertion. The behaviors of Trp and Tyr were dramatic (Figure 5c). When placed centrally, they strongly reduced membrane insertion, but they became much less unfavorable as they were moved apart. Indeed, Trp was as favorable as Leu when placed in the outermost positions (Figure 5c). The position dependence of Phe was different from that of Trp and Tyr (Figure 5c), because Phe does not have a strong interfacial preference in MPs (69, 71). The wave-like pattern observed for the Phe pair-scan is a result of variations in the hydrophobic moment (amphiphilicity) of the helices (32). These results provided further evidence supporting the idea that protein-lipid interactions are central to the recognition of TM helices by the translocon. Prediction of Transmembrane Helices The strong position dependence of Gaa app meant that the base biological hydrophobicity scale would be of limited value for predicting TM helices by simple hydropathy plot methods; accurate predictions require accounting for the position dependence of Gaa app . In a recent study, Hessa et al. (33) carried out a comprehensive examination of the position ANRV343-BB37-02 ARI 24 April 2008 15:5 dependence of Gaa app . In addition, they determined how the overall length of the Hsegment affected Gapp . The data enabled them to derive a simple expression for calculating the expected Gapp for H-segments given the amino acid sequence and overall length: pred Gapp = l i=1 2 Gaa(i) app + c 0 μ + c 1 + c 2 l + c 3 l , helices by the translocon, and support models based on a partitioning of the TM helices between the Sec61 translocon and the surrounding lipid. The details of the partitioning process remain to be determined, but presumably the open state of the translocon is a highly dynamic one that permits rapid sampling of the translocon-bilayer interface by the translocating polypeptide. 1. aa(i) Gapp where l is the length of the segment, is the matrix element giving the contribution from amino acid aa in position i, μ is the hydrophobic moment, c0 is the weight parameter for the hydrophobic moment, and the terms c1 +c2 l+c3 l 2 account for the dependence of Gapp on segment length. The optimized matrix was derived by minimizing the sum of the squared difference. A web server for calculating Gapp profiles across a protein sequence is available at http://www.cbr.su.se/DGpred/. pred Distributions of Gapp values obtained for mammalian secreted proteins as well as single- and multi-spanning MPs are shown in pred Figure 5d. The overlap between the Gapp distributions for the single-spanning TM proteins and the secreted proteins is small, and the two distributions cross close to the zeropoint on the scale defined by the experimental analysis of the designed H-segments. A surprisingly large fraction (25%) of the TM helices in the multi-spanning MPs of known pred three-dimensional structure have Gapp > −1 0 kcal mol . Such segments would presumably be only inefficiently recognized as TM helices by the translocon if they were the only hydrophobic segment in a protein. This observation suggests that TM helices in multispanning MPs may depend on interactions with neighboring TM helices for proper partitioning into the membrane. Indeed, a number of such cases have been described in the literature (66), though their overall incidence has been unclear. The results of these studies by Hessa et al. (32–34) suggest that direct protein-lipid interactions are essential for the recognition of TM Membrane Insertion of Multi-Spanning Proteins How does the Sec61 translocon handle proteins with multiple TM helices? The most revealing study published so far focused on the 6TM protein aquaporin 4 (65). By an extensive analysis using site-specific cross-linkers introduced into each of the TM helices, the authors arrived at a detailed picture of when during biosynthesis each TM helix exits the translocon and enters into the lipid bilayer. In general, the helices were observed to follow each other into the membrane in a strict N- to C-terminal succession. Certain helices, however, would first completely exit the translocon only to revisit it at a later stage when a downstream helix had just entered the translocon channel. One is thus left with a picture of a dynamic translocon that allows multiple TM helices to interact with each other at early stages of membrane integration. In this way, one may envision a mechanism whereby TM helices that would not by themselves be sufficiently hydrophobic to integrate efficiently into the membrane become embedded in the progressively folding protein. Helix-Helix Interactions What kinds of interactions might underlie helix-helix association during transloconmediated membrane insertion into the lipid bilayer? It is well established that hydrogen bonding between polar residues such as Asn or Asp can drive helix-helix interactions in both detergent micelles and biological membranes (13, 26, 89, 90) and can facilitate the www.annualreviews.org • Translocons and Transmembrane Helices 33 ANRV343-BB37-02 ARI 24 April 2008 15:5 formation of helical hairpins during translocon-mediated insertion (31). MeindlBeinker et al. recently examined (51) whether and to what extent interhelix hydrogen bonding could drive the process of translocon-mediated TM helix insertion a GGPG Arg itself, and whether the separation between the two helices within the sequence may influence any such interaction. To address these questions in a quantitative way, they extended the systematic approach established by Hessa et al. (32) to study the effects b GGPG 0.5 S4 helix ΔGapp (kcal mol-1) Arginine 0.0 4 6 8 10 12 14 16 18 20 22 24 -0.5 10 Å -1.0 -1.5 = Arg positions in S4 helix -2.0 Position Xaa ΔGapp (kcal mol-1) GGPG Xaa d GGPG 1.2 Tyr (2Y, 3L, 14A) 1.0 0.8 Phe (2F, 1L, 16A) 0.6 Trp (2W, 2L, 15A) 0.4 0.2 0.0 2 4 6 8 10 12 14 -0.2 0 -0.4 4L, 15A -0.6 -0.8 0.3 16 18 Frequency c Separation Single-span TM Multi-span TM Cytoplasmic Secreted 0.2 0.1 0.0 -10 -8 -6 -4 -2 pred ΔGapp 0 2 (kcal 4 mol-1) P2 G2 e Translocated G1 G1 Inserted ER Lumen H1 f H H2' H I II ER Lumen H1 H2' H2' D D H H2' D D D D Cytoplasm Cytoplasm G2 P1 P1 34 White (II) ΔGaaapp >>0 · 2D/17L (I) ΔΔGapp = ΔΔGapp -ΔΔGapp P2 ΔGaaapp <<0 19L von Heijne H 6 8 10 ANRV343-BB37-02 ARI 24 April 2008 15:5 of mutual helix-helix interactions on the efficiency of membrane insertion, using the scheme shown in Figure 5e. The experiments (51) yielded several important results (Figure 5f ). First, different Asn- or Asp-containing H2 sequences did not affect the insertion of a purely hydrophobic H-segment. Furthermore, little effect was seen when a signal peptidase cleavage site was introduced in H2 , or even when the entire H1-H2 region was replaced by the signal peptide from preprolactin. The H2 sequence thus had little influence on Gapp when the H-segment was composed only of hydrophobic residues (cf. Figure 5f, I). Second, by analyzing model protein constructs in which zero, one, or two Asn or Asp residues were placed in two neighboring hydrophobic segments (H2 and H), it was found that Gapp of a marginally hydrophobic H-segment was significantly reduced only if both the H2 -segment and the H-segment contained two Asn or two Asp residues (Figure 5f, II) with a spacing of three, but not one or five, residues (i.e., when they are spaced one helical turn apart in both H2 - and H-segments). These results suggest that interhelix hydrogen bonds can form during Sec61 translocon-assisted insertion, and that the H2 -segment remains in ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 5 Position dependence of Gaa app and helix-helix interactions in membrane protein assembly. (a) Scan of a single Arg residue across H-segments of composition 1R/6L/12A. The position of the Arg in the 19-residue hydrophobic stretch is shown on the x-axis. Orange circles indicate the locations of the Arg in the KvAP S4 helix. Data are replotted from Reference 34. This plot reveals the strong position Arg dependence of Gapp . Lys, Asp, and Glu residues show a similar position dependence. (b) Molecular dynamics simulation of a model S4 voltage-sensor peptide (GGPG-LGLFRLVRLLRFLRILLIIGPGG) in a palmitoyloleoylphosphatidylcholine bilayer. (Left) Cut-away view of the simulation system, showing bilayer distortion around the peptide and the contacts between phosphate groups, water molecules, and Arg guanidinium groups. (Right) This space-filling representation of the hydrophilic neighborhood of the S4 helix, represented as Connolly surfaces, reveals a 10 Å gap that is never occupied by the Arg guanidinium groups because of snorkeling to the bilayer interface. Red, water; yellow, phosphocholine headgroups; teal, acyl chains; white, GGPG. . .GPGG flanks; silver, non-Arg S4 residues; dark blue, guanidinium groups. Images modified from Reference 24 with permission. (c) H-segment pair-scans for Phe, Trp, and Tyr residues in which pairs of residues are moved symmetrically toward the N and C termini of the sequence. The compositions of the H-segments are indicated. The dashed line indicates the Gapp value for the 4L/15A H-segment. This shows that the Trp residues have the same apparent free energy as Leu when placed near the ends of the H-segment. Data pred are replotted from Reference 32. (d ) Distributions of Gapp values in cytoplasmic, secreted, and transmembrane (TM) proteins were computed from Equation 1. The 17- to 33-residue segment with pred lowest Gapp was identified in 670 cytoplasmic ( green), 1012 secreted (gray), and 349 single-spanning pred TM proteins (excluding signal peptides; blue), and the 17- to 33-residue segment with lowest Gapp within each annotated helix (plus 10 residues on either side) was identified in 508 TM helices from multi-spanning TM proteins of known three-dimensional structure ( purple ). Data points show the pred relative frequency of proteins with Gapp within ± 0.5 kcal mol−1 of the value given by the x-axis. Data are replotted from Reference 33. (e) In order to examine helix-helix interactions driven by hydrogen bonding, the native Lep H2-segment was replaced by an H2 -segment of the general composition L19-n Nn or L19-n Dn (n = 0, 1, or 2), and a 19-residue H-segment containing one or two Asn or Asp residues was inserted into the P2 domain. Redrawn from Reference 51. ( f ) To measure the effect of Aspor Asn-mediated interactions between the H2 - and H-segments, two constructs were compared for each H-segment: one with a uniformly hydrophobic 19-Leu H2 -segment (I) and one with an H2 -segment in which one or two of the Leu residues were replaced by Asp or Asn residues (II). The interaction free energy is expressed as the difference (Gapp ) in the apparent free energy of insertion of the H-segment between the two constructs. In the example shown, the H-segment contains two Asp residues and the (I ) appropriate number of Leu and Ala residues to make Gapp ≈ 0 kcal mol−1 . Redrawn from Reference 51. www.annualreviews.org • Translocons and Transmembrane Helices 35 ANRV343-BB37-02 ARI 24 April 2008 15:5 close proximity to the translocon to offer its hydrogen-bond donor and acceptor sites to the incoming H-segment even when the intervening loop is 150 residues long (30, 53, 65). THE BIOLOGY-PHYSICS NEXUS The Gapp measurements of Hessa et al. (32–34) are fully consistent with the simplest model one can propose for how TM helices are recognized by the ribosome-translocon machinery: Helices are somehow allowed to partition into the surrounding lipid bilayer because of the free energy of interaction between the TM segment and the lipid. This would explain the correspondence between the biological hydrophobicity scale and biophysical scales like that of WW, and it would explain why the positional variations in Gapp for residues such as Arg, Trp, Tyr, Phe, and Gly (32, 34) match the statistical distribution of these residues across the membrane in the high-resolution X-ray structures (69). The data at hand thus speak strongly in favor of direct protein-lipid interactions as the main driving force for the integration of single TM helices, although the translocon may affect the ability of pairs or higher-order assemblages of TM helices to interact among themselves before partitioning into the bilayer (33, 51). Although much remains to be done in order to fully understand the results obtained with the Sec61 translocon system, the H1 and H2 TM helices present in the model protein (Figure 5e) do not seem to affect the results in any significant way, as they can be replaced by a cleavable signal peptide with little effect on the measured Gapp values (51). Moreover, position-specific contributions to Gapp obtained by single-scans of a charged or polar residue along an Hsegment predict Gapp values for H-segments using symmetrical pair-scans, or even natural TM helices with multiple charged residues 36 White · von Heijne within ∼1 kcal mol−1 (32, 34). This finding suggests that vertical sliding of the Hsegments used in the derivation of the biological hydrophobicity scale is not a serious problem. This is probably not the whole story, however. Many polar and charged residues, Arg included, have rather long and flexible side chains, making it possible for them to snorkel toward the lipid-water interface region. At the same time, lipid molecules located close to a TM helix can adapt to the presence of polar residues, and water molecules can help solvate polar groups located well within the bilayer plane (17, 24, 39). One upshot of this dynamic picture of protein-lipid interactions is that Gapp profiles, such as the one shown in Figure 5a, most likely do not provide an accurate representation of the free-energy profile for moving a charged residue all the way across a membrane (as opposed to inserting it sideways from the translocon as part of a TM helix). Presumably, if a helical peptide is pulled across a lipid bilayer, there is a substantial freeenergy barrier (not seen in the Gapp profile) when a charged residue has to flip its direction of snorkeling across the 10 Å hydrophobic gap (Figure 5b) from one membrane surface toward the other (17). Seen from this perspective, one may regard the translocon as a device designed to lower the activation barrier for translocation of polar and charged residues across the membrane. It does so by providing an aqueous channel while making it possible for consecutive segments of the nascent polypeptide to make lateral excursions from the channel in order to test whether the free energy of membrane insertion is favorable. Despite these caveats, it seems likely that the biological hydrophobicity scale is a good measure of the energetics of protein-lipid interactions in the true biological context, and as such will help us define the sequence determinants of membrane-protein assembly much more precisely than has been possible so far. ANRV343-BB37-02 ARI 24 April 2008 15:5 SUMMARY POINTS 1. MPs are in thermodynamic equilibrium with the cell membrane’s lipid bilayer, which means that the stability and three-dimensional structure of MPs are ultimately determined by lipid-protein physical chemistry. 2. α-Helical MPs are identified during translation on the ribosome by the signal recognition particle that initiates docking of the ribosome to the membrane-embedded multi-protein translocon complex. 3. Elongating polypeptides from the ribosome pass through a translocon TM channel within the translocon complex. 4. The translocon’s U-shaped structure allows diversion of TM helices sideways into the lipid bilayer. 5. The diversion of the helices into the bilayer appears fundamentally to be a physicochemical partitioning process between translocon and bilayer. 6. The partitioning process can be described quantitatively by apparent free energies that serve as a code for the selection of TM helices by the translocon working in concert with the lipid bilayer. FUTURE ISSUES 1. Much more structural information about translocons and translocon complexes is needed, especially an atomic-resolution structure of a translocon engaged in polypeptide secretion. 2. Although there is a clear connection between the physical chemistry of lipid-protein interactions and selection of TM helices by the translocon, a quantitative molecular description of the empirical apparent free energies of the translocon’s selection code is needed. 3. The molecular basis for the translocon-assisted assembly of multi-spanning MPs needs to be established. DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS This research was supported by grants from the Swedish Foundation for Strategic Research, the Marianne and Marcus Wallenberg Foundation, the Swedish Cancer Foundation, the Swedish Research Council, and the European Commission (BioSapiens) to GvH, and by grants from the National Institute of General Medical Sciences to SHW. We thank Michael Myers for editorial assistance and the TEMPO group at U.C. Irvine for useful discussions. www.annualreviews.org • Translocons and Transmembrane Helices 37 ANRV343-BB37-02 ARI 24 April 2008 15:5 LITERATURE CITED 1. Allen SJ, Curran AR, Templer RH, Meijberg W, Booth PJ. 2004. Folding kinetics of an a-helical membrane protein in phospholipid bilayer vesicles. J. Mol. Biol. 342:1279–91 2. 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