Principles of membrane protein structure

PR! NC! PLES OF MEMB RAN E PROTEIN STRUCTURE

M. S. P. Sansom and lan D. Kerr

I. II.

IlI.

IV.

VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of M e m b r a n e Proteins . . . . . . . . . . . . . . . . . . . . . . . A. Integral and Peripheral M e m b r a n e Proteins . . . . . . . . . . . . . . . . B. Single T M Helices . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. A l l - ~ Topologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. All-~ Topologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. " M i x e d " Topologies . . . . . . . . . . . . . . . . . . . . . . . . . . . All-c~ IMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Experimentally D e t e r m i n e d Structures . . . . . . . . . . . . . . . . . . . B. Topology Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Structure-Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . All- I] IMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Experimentally D e t e r m i n e d Structures . . . . . . . . . . . . . . . . . . . B. Related Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

30 31 31 32 33 34 34 34 34 41 48 60 60 63

~13 IMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nicotinic Acetylcholine Receptor . . . . . . . . . . . . . . . . . . . . . .

65 65

Biomembranes Volume 1, pages 29-78. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-658-4 29

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M.S.P. SANSOM and IAN D. KERR

B. Voltage-GatedIon Channels . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

69 71 72 72

INTRODUCTION

Why attempt to elucidate principles of membrane protein structure? First, such principles help to clarify the relationship between the structures of membrane proteins and their diverse functions. Second, an understanding of such principles may enable accurate prediction of membrane protein structures. Sequencing membrane proteins has become relatively straightforward, whereas determination of their three-dimensional (3D) structures remains slow and difficult. Consequently, this predictive aspect is of considerable importance. The following discussion focuses on the structure of integral membrane proteins (IMPs), and in particular, on their transmembrane (TM) domains. Many membrane proteins also possess extensive extramembranous domains. Available evidence suggests the folding of such domains follows the same principles as for soluble proteins. There already exists an extensive literature concerning the latter, to which the interested reader is referred (Schulz and Schirmer, 1979; Chothia, 1984; Branden and Tooze, 1991). High-resolution three-dimensional structures are known for only a handful of integral membrane proteins. It, therefore, may seem premature to discuss principles of membrane protein structure. However, as a result of detailed examination of known 3D structures, and of computational analysis of numerous membrane protein sequences, general principles have started to emerge. The database of high-resolution structures of IMPs is summarized in Table 1. Both X-ray diffraction from three-dimensional crystals (Michel, 1991) and electron diffraction from two-dimensional (2D) crystals (Kuhlbrandt, 1992) have been used to determine these 3D structures. In addition to true IMPs, crystallographic and nuclear magnetic resonance (NMR) structures are known for several amphipathic channel-forming peptides (e.g., melittin, Terwilliger and Eisenberg, 1982; alamethicin, Fox and Richards, 1982; 8-toxin, Tappin et al., 1988) which may be considered as models of helical TM domains (Sansom, 1991, 1993a,b). High-resolution structures have also been determined for a number of membrane-active toxins which provide insights into membrane protein structures (Li, 1992; Parker and Pattus, 1993). Together, these three classes of structure provide sufficient data to enable definition of some general principles. Despite the limited size of the experimental database, prediction of membrane protein structure is more feasible than structure-prediction of soluble proteins. The 2D nature of a bilayer places considerable constraints on possible 3D structures, allowing experimental and theoretical analysis of transbilayer topologies of

Principles of Membrane Protein Structure

Table 1. Protein

Membrane Protein Structures

Method

bacteriorhodopsin halorhodopsin rhodopsin photosynthetic reaction center

31

Resolution ,~a ~, (2D) ~, (2D) ]k

Reference

EM EM EM X-ray

3.5 6.0 9.0 2.3

Henderson et al. (1990) Havelka et al. (1993) Schertler et al. (1993b) Deisenhofer et al. (1985)

X-ray

3.1 ]k

Allen et al. (1987)

EM X-ray X-ray X-ray X-ray EM

3.4 ,~ 6.0/k 1.8/~ 2.0 ,~, 3.0/k 9.0/k

Ktihlbrandt et al. (1994) Krauss et al. (1993) Weiss et al. (1991b) Kreusch et al. (1994) Cowan et al. (1992) Unwin (1993)

(Rhodopseudomonas viridis) photosynthetic reaction center

(Rhodobacter sphaeroides) light harvesting complex photosystem I (Synechococcus) porin (Rhodobacter capsulatus) porin (Rhodopseudomonas blastica) porins OmpF and PhoE (E. coli) nicotinic acetylcholine receptor

Note: a3.5~, resolution in ~,; 10 ,~ resolution in z.

polypeptide chains. Thus, it is possible to generate credible models of IMP structures by combining analysis of amino acid sequences with experimentally derived structural constraints. In the following discussion, the relationship between experimental structures, general principles and structure-prediction will be examined and evaluated.

il. CLASSIFICATION OF MEMBRANE PROTEINS A. Integral and Peripheral Membrane Proteins There are two major types of membrane proteinsmintegral and peripheral. Integral proteins interact strongly with membranes as a result of their polypeptide chains spanning the bilayer one or more times, whereas peripheral proteins interact more weakly with the membrane surface. Peripheral proteins may form electrostatic interactions with phospholipid headgroups or may bind to integral proteins. Peripheral proteins are thought to obey the same structural principles as soluble proteins, and so will not be discussed further. The key structural feature of IMPs is that their polypeptide chains span lipid bilayers, thus excluding those regions of the chains from contact with water molecules. This has two structural consequences: (a) membrane-spanning regions of polypeptide chains adopt a defined secondary structure, often (~-helical; and (b) membrane-spanning sequences are predominantly hydrophobic. If a membranespanning polypeptide was to adopt a random coil conformation, exclusion of water molecules from the bilayer would result in unfulfilled H-bonds, at an energetic cost

32

M.S.P. SANSOM and IAN D. KERR

A

R /)

B

Figure 1. Schematic diagrams of possible transbilayer topologies of IMPs. Cylinders represent (x-helices and arrows represent I~-strands. A represents those membrane proteins which possess a single TM helix. B represents all-c~ IMPs, with multiple a-helices crossing the bilayer. C shows an all-13 topology, and D a "mixed" ~13 topology, with both c~-helical and 13-sheetTM domains.

of ca. +6 kcal/mol/H-bond. Thus, simple thermodynamic arguments favor defined secondary structures for membrane-spanning chains. The first level of characterization of the structure of an IMP is thus to determine the location in the sequence of the membrane-spanning regions, to establish their secondary structure, and to define how the membrane-spanning elements are threaded back and forth across the bilayer. This is referred to as the transbilayer topology of the polypeptide chain. The requirement for hydrophobic side-chains to interact with the bilayer core aids in prediction of such topologies from amino acid sequences.

B. Single TM Helices The simplest structure of an IMP is a single TM helix (Figure 1A). A 20 residue o~-helix (1.5 A/residue) will span a lipid bilayer of thickness 30 A. Longer or shorter

33

Principles of Membrane Protein Structure

helices may form TM domains if local distortion of the bilayer occurs (Mouritsen and Bloom, 1984, 1993). Side-chains of a single TM helix will be predominantly hydrophobic. Such IMPs may posses extensive globular domains on either one or both faces of the bilayer. The TM domain may simply act as an "anchor" to localize a globular domain in a membrane, or may have a more active role. For example, it has been suggested that intramembrane helix-helix interactions may play a role in cellular signaling (Bormann and Engelmann, 1992). The TM helices of glycophorin, an intensively studied single TM helix protein from erythrocytes, are capable of sequence-specific aggregation within the lipid bilayer phase (Bormann et al., 1989; Lemmon et al., 1992). Thus, even though a single TM helix exhibits a simple topology, possible complexities resulting from oligomerization must be considered. For example, Popot and de Vitry (1990) have suggested that inner mitochondrial and thylakoid membranes contain large numbers of single TM helix IMPs which microassemble within the bilayer to form functional oligomers. Such assemblies will be considered alongside more complex all-or IMPs.

C. All-ix Topologies Many IMPs exhibit more complex topologies, their polypeptide chain crossing the bilayer several times. In such molecules, a considerable fraction of the mass of the protein may be located within the bilayer. Arguably, the most common class of such IMPs are those with an all-tx topology (Figure 1B; Table 2), in which several TM helices span the bilayer. This class of IMPs may be extended to include microassemblies of single TM helix proteins (see above), channel-forming peptides (CFPs), and membrane-active toxins which form transbilayer pores by intramembranous association of TM helices to generate parallel bundles. In all-or IMPs, the TM helices are again generally hydrophobic. However, particularly in ion channel and transport proteins, amphipathic helices may occur which associate to form

Table 2. Classification of Integral Membrane Proteins Class

Description

all-o~

bundle of TM u-helices

all-13 ~13

antiparallel l-barrel o~-helicesand 13-strandsin the sameTM domain

Members

bacteriorhodopsin halorhodopsin rhodopsin photosyntheticreaction centers light harvestingcomplex photosystemI porins nicotinicacetylcholinereceptor K+ channels?

34

M.S.P. SANSOM and IAN D. KERR

bundles in which the hydrophilic surfaces of the helices line a central pore or binding site.

D. AII-~ Topologies Initially, it was suggested that all IMPs might contain predominantly tx-helical TM domains (see e.g., Capaldi, 1982). However, since the determination of high-resolution structures of several porins, it has become evident that a [5-sheet may also yield a stable TM fold (Figure 1C; Table 2). In order to avoid unfulfilled H-bonds at its edges, the sheet is folded back on itself to form a transbilayer [5-barrel. It remains to be seen whether this fold will be found in IMPs other than porins and related proteins.

E. "Mixed" Topologies More recently, two IMP structures have been suggested to contain a "mixed" ct/13 topology. Significantly, both of these structures are for ion channels, possessing a central transbilayer pore. For the nicotinic acetylcholine receptor, it is suggested that a central bundle of TM helices is surrounded by a hydrophobic 13-barrel. For voltage-gated channels, it is suggested that the central pore is formed by a [5-barrel which is, in turn, surrounded by an outer bundle of TM helices. In both cases, the functional protein is an oligomer, with o~-helices and [5-strands donated by each of the subunits. Overall, IMP topologies may be divided into three classes: ct, 13, and ct/13, with the first of these apparently the most widespread. In all cases, membrane-spanning segments are either hydrophobic or amphipathic. Furthermore, intramembranous association of TM domains contributed by different subunits may occur.

i11. ALL-ix IMPs In this section we first discuss all-or IMPs whose structures have been determined at atomic or near-atomic resolution. In particular, we focus on general rules which may be derived from these structures. We then describe how such structural principles may be employed in prediction of transbilayer topologies of all-or IMPs. Finally, we assess attempts to predict the intramembranous packing of TM helices.

A. Experimentally Determined Structures Two high-resolution structures are availablemthose of bacteriorhodopsin (bR) and the photosynthetic reaction center (PS/RC). Lower resolution structures are available for halorhodopsin (hR), bovine rhodopsin, a photosynthetic light harvesting complex (LHC), and photosystem I (PS-I). The number of TM helices observed

Principles of Membrane Protein Structure

35

in these proteins are: bR, hR and rhodopsin, 7; PS/PC, 11; LHC, 3; and PS-I, 21. Thus, the two high-resolution structures contain TM helix bundles of an intermediate level of complexity.

Bacteriorhodopsin The seven-helix bundle structure (Figure 2) of bR was first determined at 7 resolution by Henderson and Unwin (1975). This represented a significant breakthrough in the study of IMP structure. The more recent determination of the structure of bR at near-atomic resolution (Henderson et al., 1990) provides much valuable information concerning the packing of TM helices within IMPs. The basic structure is a compact bundle of seven helices (A to G; Figure 2), which lie approximately perpendicular to the plane of the bilayer and surround an elongated central pocket containing the chromophore retinal (covalently linked to Lys-216 of helix G), and a proton-conducting "channel" to the extracellular surface. Viewed from the extracellular face of the membrane (corresponding to the N-terminus of the polypeptide chain) helices A to G are in a counterclockwise order. The anisotropy of the resolution (3.5 A in xy, 10 A in z, where xy is the plane of the bilayer) results in poorer definition of interhelix loops than of TM helices. The development of 3D crystals of bR which are suitable for X-ray diffraction studies (Schertler et al., 1993a) is expected to overcome the latter problem. The lengths of the TM helices and interhelix loops are given in Table 3. A mean helix length of 23 residues (ca. 35 ,~) is sufficient to completely span the hydrophobic region of a lipid bilayer. The interhelix loops (mean length = 10 residues) are all quite short. Thus, most of the polypeptide chain lies within or very close to the bilayer. Helices B, C and F contain proline residues, resulting in kinked helices. The kink angles are: B, 6.6~ C, 17.3~ and F, 30.2 ~ As discussed below, intrahelical prolines occur with a relatively high frequency in certain classes of IMP, and may play important structural and/or functional roles. Although the TM helices are largely hydrophobic, they contain some polar and charged residues, most of which point toward the interior of the bundle. Tryptophan residues are present at the N-termini of helices A, C, and E, i.e., close to the presumed position of the bilayer/water interface (Schiffer et al., 1992). The packing of helices within bR is summarized in Table 4. All adjacent helix pairs are antiparallel, except for the G-A pair which "closes" the bundle. The mean helix crossing angle for the antiparallel pairs is f~ = - 168 ~ which is consistent with class 3-4 "ridges-in-grooves" helix packing as defined by Chothia et al. (1981). Overall, the shape of the bundle is that of a distorted left-handed supercoil. Interhelix separations are also typical of those found for globular proteins (Reddy and Blundell, 1993). Thus bR appears to obey similar rules of helix-helix interactions to those which apply for globular proteins.

A

B

C

D

B

Figure 2. Bacteriorhodopsin. Two views of the seven-helix bundle are shown. A is down a perpendicular to the plane of the bilayer, showing the s-carbon traces of the seven TM helices. The same helices are shown in "ribbon" format in B, in a view approximately perpendicular to that in A. The individual helices and the N- and C-termini of the polypeptide chain are labeled. Interhelix loops are omitted from both diagrams. (Coordinates taken from entry 1BRD of the Brookhaven PDB; Bernstein et al., 1977). 36

Table 3. AII-a IMPS: TM Helices and Loops Protein bR

Helix

Length

A B C D E F G

23 25 21 20 21 25 23

mean + SD PS/RC

Proline.ga

L1 L2 L3 L4 L5 M1 M2 M3 M4 M5 H1 mean + SD

P 13 P12

P20

Loop A-B B-C C-D D-E E-F F-G

Length 5 17 7 9 9 11

23 + 2

mean + SD

10 + 4

21 29 26 30 27 27 30 26 29 27 26

P4, P 10 P2

L l-L2 L2-L3 L3-L4 L4--L5

28 2 29 25

P22 P2

M l-M2 M2-M3 M3-M4 M4-M5

31 2 29 33

mean + SD

22 + 12

27 + 2

Note: aNumber indicates position of the proline within the TM helix.

Table 4. Packingof TM Helices Protein bR

Helix Pair A-B B-C C-D D-E E-F F-G G-A mean o + SD

PS/RC

LA-LB LB-LC LC-MD MD-LE LE-LD MA-MB MB-MC MC-LD LD-ME ME-MD mean + SD

f~ (o)a

R (it)

-155 -167 +173 -160 -170 -170 +8

8.5 9.7 9.7 9.3 11.5 10.8 10.7

-1680 + 10

10.0 + 0 . 9

-154 -149 -124 -127 -157 - 160 -157 -128 -128 -163

7.9 10.3 11.5 10.4 9.2 8.1 10.4 10.9 9.8 8.8

-145 + 15

9.7 __+1.1

Notes: a~ is the helix crossing-angle and R is the interaxial separation at the closest contact of the helices. For an exactly parallel helix pair, f~ = 0~ for an exactly antiparallel pair, f2 = + 180~ ~ the parallel helix pair (i.e., G-A).

37

38

M.S.P. SANSOM and IAN D. KERR

Halorhodopsin The structure of halorhodopsin, a light driven C1-pump with 32% sequence identity with bR, has been determined at 6/k resolution in projection perpendicular to the plane of the membrane (Havelka et al., 1993). The projection-structure ofhR is almost identical to that ofbR at the same resolution, hR contains seven TM helices which have similar tilt angles, relative to the bilayer normal, to those of bR. Thus the strong sequence similarity between the two proteins results in a common transbilayer architecture.

Rhodopsin A 9 ,~ resolution projection-structure has been determined for bovine rhodopsin (348 residues; Schertler et al., 1993b). As predicted (see below), this reveals some structural homologies with bR in that there are seven TM helices in a closed bundle. However, whereas in bR three of the helices are approximately perpendicular to the bilayer (B, C and Dmsee Figure 2) and four are tilted, in rhodopsin the opposite is true, with four helices perpendicular to the bilayer while three are tilted. Furthermore, the rhodopsin bundle (in cross-section) is less elongated than that of bR. This demonstrates that even when two proteins share a common TM topology, significant differences may occur in the packing of their helices.

Photosynthetic Reaction Center Photosynthetic reaction center (PS/RC) structures have been determined for two closely related species (Deisenhofer et al., 1985; Deisenhofer and Michel, 1989; Allen et al., 1987). The TM helix bundles are similar in both structures and so the following discussion will be concerned with the R. viridis PS/RC. The R. viridis PS/RC is made up of four subunits: L, M, H, and a cytochrome (Figure 3). The latter is a peripheral-membrane protein and so will not be considered further. Indeed, the principal difference between the R. viridis and R. sphaeroides complexes is the absence of a cytochrome subunit from the latter. The extramembranous domain of the H subunit is on the cytoplasmic face of the membrane. Thus the IMP may be considered as composed of three subunits L, M, and H. Within the IMP, there are also four bacteriochlorophylls, two bacteriopheophytins, a quinone, and an Fe 2+ ion. Subunits L (273 residues) and M (323 residues) have very similar folds, each containing five TM helices. These are packed together in the order ABCED (Figure 3b). The H subunit (259 residues) is mainly cytoplasmic, and has a single TM helix. Thus, there are 11 TM helices in all. These form a flattened, open-ended bundle, distinct from the closed bundle of bR, with an approximate twofold axis relating the L and M subunits. The helices are tilted somewhat relative to the plane of the bilayer.

Principles of Membrane Protein Structure

39

A

Cyt

~i!ii~!ii~iil

i i i i~,ii i i i!i i il ~ii~i~i!i!!!~ii!iii

Figure 3. Rhodopseudomonas viridis photosynthetic reaction center. A shows the (x-carbon trace of the complete PS/RC molecule, viewed within the plane of the bilayer. The four subunits (H, L, M, and Cyt) and the approximate location of the lipid bilayer (shaded) are indicated. The eleven TM helices are shown in B (same view as in A) and C (viewed perpendicular to the plane of the membrane). TM helices of the L and M subunits are shown as light and dark grey ribbons respectively, with the single TM helix of the H subunit at an intermediate level of shading. (Coordinate entry 1PRC of the P D B ) . (continued)

The TM helices of PS/RC are on average somewhat longer than those of bR (Table 3). With the exception of the short LB-LC and MB-MC loops, the interhelix loops are much longer than those of bR, reflecting the greater proportion of the polypeptide chain outside the bilayer region, intrahelical prolines are present, resulting in kinks in helices LC and MC. Helices LE and ME also have pronounced kinks, although they do not contain prolines.

40

M.S.P. SANSOM and IAN D. KERR

B

C

A

Figure 3. (continued)

Packing of the TM helices (Table 4) differs slightly from that of bR in that the crossing angles deviate somewhat more from-180 ~ (mean D = -145~ Helices LD, LE, MD, and ME form a classic four-helix bundle motif (Weber and Salemme, 1980) at the center of the molecule. The crossing angles between LD and ME (and MD and LE) are close to those for class 1-4 "ridges-in-grooves" packing. Helixhelix separations are similar to those for bR.

Principles of Membrane Protein Structure

41

Overall, the PS/RC provides an example of a more extended, flatter bundle of TM helices. It also demonstrates how TM helices from different subunits may contribute to such bundles. Interactions between helices from different subunits do not differ in essence from those between helices donated by the same subunit. This has implications with respect to methods of predicting packing of TM helices.

Light HarvestingComplex The light harvesting complex (LHC) associated with photosystem II (PSII) from chloroplast membranes has a molecular weight of 25 kDa and contains 15 chlorophyll molecules. The structure of LHC (Ktihlbrandt and Wang, 1991; Ktihlbrandt et al., 1994) reveals three TM helices. Helices A and B are ca. 30 residues long and form a central pair on an approximate twofold axis. They are tilted at angles of ca. 25 to 30 ~ to the bilayer normal. Helix C sits to one side of the A-B pair, is ca. 21 residues long, and is tilted at ca. 10 ~ to the bilayer normal. The order of the helices in the sequence is BCA. Thus, A and B are approximately parallel. They pack with an interhelical separation of R ca. 9 A and a helix-crossing angle of f~ ca. +60 ~ This unusual helix-crossing angle is a result of helix-chromophore interactions. Two carotenoid molecules sit in the grooves of the supercoil defined by helices A and B and thus play a structural role.

Photosystem-i The 6 ,~ resolution X-ray structure for PS-I from Synechococcus (Krauss et al., 1993) provides an example of a large and complex all-c~ IMP. PS-I is made up of two large subunits, A and B (each ca. 83 kDa) and five small subunits (3-15 kDa), in addition to 45 chlorophyll a molecules, and three 4 Fe-4S clusters. There are 28 o~-helices, of which seven lie approximately parallel to the bilayer, and 21 are TM helices. The latter are tilted at angles ranging from 3 ~ to 30 ~ relative to the bilayer normal. The overall TM helix bundle is elongated, with an approximate twofold axis relating helices a to h of subunit A to helices a' to h' of subunit B. At 6 / k resolution, it is not possible to determine the polarity of the helices or to trace the interconnecting loops. However, it is clear that when extended to higher resolution, the structure of PS-I will greatly increase our knowledge of TM helix packing.

B. Topology Prediction In addition to the experimental structures discussed above, there have been several attempts to develop methods for prediction of TM topologies of all-or IMPs. In the following section we review the theoretical background and implementation of such methods, and illustrate their application to bR and related proteins. We also briefly review some methods for experimental evaluation of predicted topologies.

42

M.S.P. SANSOM and IAN D. KERR

Hydrophobicity Scales The underlying theory of topology predictions for all-(x IMPs is that TM segments consist of hydrophobic (x-helices 20-30 residues long, i.e., sufficiently long to span at least the hydrophobic core of a lipid bilayer. Engelman et al. (1986) analyzed the thermodynamics of insertion of an Ala20 helix into a lipid bilayer. U(aq) ~

~+40

0

H(aq)

~-30

(l)

-70 U(lip) ~ n(lip) where U is the unfolded polypeptide chain and H is the (x-helical conformation, aq and lip are the aqueous and lipid bilayer environments, and the figures are AG values in kcal/mol. Thus, transfer of a preformed Ala20 helix from water to a bilayer is favored by ca.-30 kcal/mol. Ala20 is a relatively hydrophobic helix. To determine the locations of TM helices within an amino acid sequence one needs to estimate the corresponding transfer free energies for helices with different sequences. There have been numerous proposed sets of values of single amino acid hydrophobicities, i.e., hydrophobicity scales (Cornette et al., 1987). The derivation of these has ranged from physico-chemical measurements to statistical analyses of known (globular) protein structures. Four such scales are shown in Table 5. The KD (Kyte and Doolittle, 1982) scale is based on a combination of statistical data on distribution of amino acids between surface-exposed and buried locations in globular protein structures, and of water-vapor partition coefficients for amino acid analogues. The EIS scale (Eisenberg et al., 1984) is a consensus scale, derived from five other scales and normalized to a mean of zero and a standard deviation of one. The GES scale (Engelman et al., 1986) was obtained via calculation of transfer free energies (see above) using hydrophobic terms derived from accessible surface area calculations, and hydrophilic terms derived from calculations of Born and H-bonding energies. Finally, the VH (von Heijne, 1992) scale is derived from results of topology predictions and analysis of 135 TM helices from 24 bacterial IMPs. These five hydrophobicity scales show broad similarities. Positive values correspond to hydrophobic residues, negative values to hydrophilic residues. There are, however, some detailed differences in the ranking of the amino acids, particularly with respect to the values for Trp, Tyr and Pro. It is significant that the former two residues are somewhat amphipathic, and that proline plays an idiosyncratic role in TM helices (see below).

Hydrophobicity Profiles In order to locate possible TM helices within amino acid sequences, one searches for clusters of ca. 20 to 25 predominantly hydrophobic residues. Each distinct

43

Principles of Membrane Protein Structure Table5. Hydrophobicity Scales Scales Residue

EISa

Arg (R) Lys (K) Asp (D) Gin (Q) Asn (N) Glu (E) His (H) Ser (S) Thr (T) Pro (P) Tyr (Y) Cys (C) Gly (G) Ala (A) Met (M) Trp (W) Leu (L) Val (V) Phe (F) Ile (I)

-2.53 -1.50 --0.90 -0.85 -0.78 -0.74 -0.40 -0.18 -0.05 +0.12 +0.26 +0.29 +0.48 +0.62 +0.64 +0.81 +1.06 + 1.08 +1.19 +1.38

Notes:

KDb

-4.5 -3.9 -3.5 -3.5 -3.5 -3.5 -3.2 -0.8 -0.7 -1.6 -1.3 +2.5 -0.4 + 1.8 + 1.9 -0.9 +3.8 +4.2 +2.8 +4.5

GES c

-12.3 -8.8 -9.2 -4.1 -4.8 -8.2 -3.0 +0.6 + 1.2 -0.2 -0.7 +2.0 +1.0 + 1.6 +3.4 +1.9 +2.8 +2.6 +3.7 +3.1

VHa

-2.75 -3.00 -2.30 -1.81 -1.99 -2.44 -2.19 -0.12 -0.08 -0.45 -0.39 +1.81 +0.16 +0.27 +0.14 -0.88 +0.62 +0.72 +0.43 +0.97

aEisenberget al. (1984). bKyte and Doolittle (1982). CEngelman et al. (1986). avon Heijne (1992).

hydrophobic cluster is presumed to correspond to a separate T M helix. Simply plotting the hydrophobicity (hi) of each residue as a function of its position in a sequence (i) results in an extremely noisy graph which is difficult to interpret. This is overcome by smoothing the trace in order to obtain a h y d r o p h o b i c i t y p r o f i l e (Kyte and Doolittle, 1982; Engelman et al., 1986; von Heijne, 1992). Pronounced peaks in the hydrophobicity profile correspond to putative TM helices. Smoothing is achieved by averaging the hydrophobicities of e.g., 10 residues on either side of a central residue. This is achieved by multiplication by a suitable window function, i.e.: j=+n

Hi = s

wjhi+j

(2)

j--~n

where Hi is the smoothed hydrophobicity centered about residue i, h is the hydrophobicity of an individual residue and w is the window function. The window

44

M.S.P. SANSOM and IAN D. KERR

length is 2n + 1. Thus n = 10 yields a 21 residue window suitable for detection of TM helices. Differently shaped window functions may be employed. The simplest is a rectangular window, i.e., w = 1 for all 2n + 1 residues within the window. More complex windows, e.g., a trapezoidal function (von Heijne, 1992), place greater weight on central residues of the window and less on outer residues. This is intuitively attractive in that it permits occasional polar residues to occur at the ends of TM helices, i.e., in bilayer/water interfacial locations.

Application to bR and Related Proteins Hydrophobicity profiles for bacteriorhodopsin and two related proteins, halorhodopsin and bovine rhodopsin, are shown in Figure 4. These profiles were calculated using the EIS hydrophobicity scale, with a trapezoidal 21-residue window function as described by von Heijne (1992). As discussed above, hR shares 32% sequence identity with bR, and exhibits the same TM topology (Oesterhelt and Tittor, 1989; Havelka et al., 1993). The profiles for bR and hR are very similar (Figure 4a). Both show five distinct peaks for helices A to E, and a more complex double peak corresponding to helices F and G. It is interesting to note that helices F and G of bR are amphipathic (see below). By selection of a suitable cutoff level, it is possible to automatically assign at least helices A to E to positions in the bR and hR sequences. Much discussion in the literature has concerned suitable protocols for automatic assignment of TM helices on the basis ofhydrophobicity profiles. One approach (von Heijne, 1992; also see Creighton, 1993) has been to analyze distributions of hydrophobicity profile peak heights, , for TM and non-TM helices. The resultant distribution is bimodal and so allows objective assignment of a cutoff value for . Bovine rhodopsin shows little sequence homology with bR, but both hydrophobicity profile analysis and the 9/~ projection structure (see above) support a seven TM helix bundle. The hydrophobicity profile for rhodopsin (Figure 4b) exhibits seven distinct peaks, corresponding to seven TM helices. Peaks A, B, D, E, and F are quite clear, the other two more complex. This analysis permits assignment of the positions of the TM helices in rhodopsin and also in related G-protein coupled proteins (see below).

Additional Methods Two types of extension to the hydrophobicity profile method have been developed: (a) elaboration of sequence rules allowing more accurate location of hydrophobic TM helices, and (b) development of methods for identification of amphipathic TM helices. A simple extension of the hydrophobicity profile method is the so-called "positive inside" rule. This is derived from the observation that positively charged residues (Arg and Lys) are more abundant in cytoplasmic than extracellular loops

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Figure 4. Hydrophobicity profiles calculated using the von Heijne (1992) trapezoidal window algorithm and the EIS hydrophobicity scale (Eisenberg et al., 1984). The upper diagram shows profiles for bacteriorhodopsin (solid line) and halorhodopsin (broken line) superimposed. The lower diagram shows the profile for bovine rhodopsin. Positive hydrophobicity values correspond to hydrophobic regions, negative values correspond to hydrophilic regions. Experimentally determined positions of the seven TM helices (A to G) of bacteriorhodopsin are indicated above its profile. The predicted TM helices are indicated for rhodopsin.

45

46

M.S.P. SANSOM and IAN D. KERR

of all-tz IMPs. von Heijne (1992) has incorporated this rule into a predictive procedure, and demonstrated a high success rate for prediction of topologies of all-tz IMPs from bacterial membranes. A similar rule has been shown to apply to eukaryotic all-tz IMPs (Sipos and von Heijne, 1993). A second extension to the hydrophobicity profile method allows the termini of TM helices to be identified more accurately. As discussed for bR, tryptophan residues are frequently found at or near the termini of TM helices. This observation has been extended to the PS/RC by Schiffer et al. (1992). Sipos and von Heijne (1993) demonstrated that in general, Trp and Tyr residues occur with higher frequency at the termini of TM helices. This rule locates amphipathic aromatic residues in the interfacial region of the bilayer, close to polar lipid headgroups and to "penetrating" water molecules (Wiener and White, 1992). The second major extension to the hydrophobicity profile method is the development of procedures for detection of possible amphipathic TM helices. These are of particular importance in the context of channels and related transport proteins, which are thought to contain central bundles of amphipathic TM helices (Oiki et al., 1990; Sansom, 1991). Methods for detection of amphipathic helices range from simple graphical procedures (e.g., the helical wheel of Schiffer and Edmundson, 1967), to more complex sequence analysis procedures, e.g., estimation of hydrophobic moments (Eisenberg et al., 1982, 1984; Eisenberg, 1984), or Fourier analysis of sequences (Finer-Moore and Stroud, 1984). All such methods search for periodicities in sequences of residue hydrophobicities in order to detect the hydrophilic and hydrophobic faces of amphipathic helices. Perhaps the most widespread of the latter methods is the hydrophobic moment, defined by:

2

2 /1/2

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where the summation is over e.g., the 21 residues of a TM helix and where 8 - 100 ~ is the angle at which successive side-chains point away from the helix axis. The hydrophobic moment is plotted as a function of the central residue and peak values of kt correspond to the centers of putative amphipathic helices.

Evaluation of Predictions It is essential to evaluate the accuracy of TM topology predictions. There may be cases for which the prediction strategy outlined above fails. Wallace et al. (1986) provide a useful critique of the uninformed use of secondary structure prediction methods designed for globular proteins when analyzing IMPs. The simplest test of a predicted topology is to compare the fraction of the polypeptide chain predicted to occur as TM helices with experimental estimates of the secondary structure composition. Two main methods are available for estimation of the secondary structure content of IMPs" circular dichroism (CD; Johnson,

Principles of Membrane Protein Structure

47

1988) and Fourier transform infrared (FTIR; Braiman and Rothschild, 1988; Surewicz et al., 1993) spectroscopy. The results of such studies should, if nothing else, permit assignment of an IMP to the all-~ or all-13 class, and thus allow choice of an appropriate topology prediction methodology. CD has been used for some time to analyze protein secondary structures. However, when applied to membrane proteins, potential artifacts may occur, which if uncorrected can lead to erroneous estimates of secondary structure. By paying careful attention to environmental influences, such as the presence of detergents and the size of membrane vesicles, it is possible to correct for artifacts and so obtain accurate estimates of secondary structure composition (Swords and Wallace, 1993). Of course, not all of the c~-helices present in an IMP may be TM helices. However, recent developments in deconvolution of CD spectra offer the possibility of distinguishing between peripheral and TM helices in IMPs (Park et al., 1992). FTIR spectroscopy also provides estimates of the o~-helical content of IMPs, allowing distinction between all-c~ and all-I] topologies (Hafts and Chapman, 1992). It is applicable to proteins in a variety of media (Haris and Chapman, 1988), and so is well suited to investigations of membrane proteins. Overall, CD and FTIR enable the presence of a significant content of TM helices within an IMP to be detected. Furthermore, recent developments in both CD (Vogel, 1987; Huang and Wu, 1991) and IR (Frey and Tamm, 1991) spectroscopy applied to oriented planar bilayers allow the orientation of c~-helices relative to the bilayer (i.e., parallel or perpendicular) to be determined, thus providing further tests for proposed TM topologies. Spectroscopic methods estimate the overall content of TM helices within an IMP. There are also a number of techniques which permit experimental investigation of the topology of TM crossing by a polypeptide. Among the more established techniques for achieving this are limited proteolysis, chemical labeling, and the use of antibodies against synthetic peptide epitopes (reviewed by Gennis, 1989). A more recent approach, particularly applicable to analysis of bacterial IMPs, uses gene-fusion techniques (Hennessey and Broome-Smith, 1993). A reporter molecule, which exhibits location (i.e., intracellular vs. extracellular) specific phenotypes, is fused to C-terminally truncated versions of an IMP. Varying the length of the truncated IMP and determining the resultant location of the reporter molecule allows one to map the TM topology. Suitable reporter molecules include alkaline phosphatase, [~-lactamase and ~-galactosidase. For example, all three reporter molecules, when applied to the ArsB protein (the IMP subunit of a bacterial anion-transporting ATPase), yielded internally consistent results concerning the topology of this 11-TM helix IMP (Wu et al., 1992). At present, this approach has been largely confined to E. coli IMPs, but initial investigations of eukaryotic systems have been carried out. In summary, there now exist several experimental techniques for confirmation or rejection of predicted TM topologies. Such topologies, in themselves, provide a valuable conceptual framework for further analysis of membrane proteins. Hydro-

48

M.S.P. SANSOM and IAN D. KERR

phobicity profiles are routinely employed to predict TM topologies of a wide range of all-o~ IMPs. The resultant topological models may then be used to direct e.g., structure/function studies via site-directed mutagenesis.

C. Structure-Prediction

TM Helix Packing The current model for folding of all-~ IMPs is the two-stage model proposed by Popot and Engelman (1990; also see Popot, 1993). In the first stage, independently stable TM helices insert across the bilayer. In the second stage, these helices pack together, without transbilayer topological rearrangements, in order to form the tertiary structure of the intramembranous domain of the protein. Thus, TM o~-helices act as autonomous folding units. This theory has important consequences for structure-predictions, implying that simulation of packing of "preformed" TM helices may mimic the in vivo folding process. Before considering structure-prediction studies, it is valuable to review the evidence upon which this model is based. Two systems have been studied in some depth, bacteriorhodopsin and the E. coli lac permease. A number of studies have indicated that isolated TM helices adopt similar conformations to those in intact proteins, and that in vitro assembly of isolated TM helices into functional proteins may occur within a bilayer environment. The most convincing demonstration of TM helices behaving as autonomous folding units comes from NMR studies of isolated TM fragments of bR. TM helix A was prepared as a proteolytic fragment corresponding to residues 1-36 of bR, and its NMR structure determined in both methanol/chloroform and in SDS micelles (Pervushin and Arseniev, 1992). Residues 8-32 adopted an o~-helical conformation in both environments, whereas the N-terminal region was flexible. Note that in intact bR, helix A runs from residue 10 to 32. Thus, the same sequence that forms helix A in the intact structure adopts a helical conformation in an isolated peptide when the latter is present in a membrane-mimetic environment. Helix B peptide (residues 36-65, prepared synthetically with norleucine substituted for methionine) when present in SDS micelles adopted a helical conformation from residues 41 to 62 (Lomize et al., 1992). Helix B runs from residue 38 to 62 in intact bR. The peptide was kinked at Pro-50, with a kink angle of 27 ~ compared to a corresponding angle of 6.6 ~ in intact bR. Thus, the secondary structure in the intact protein was preserved in the isolated peptide, but the proline-induced kink was somewhat larger. This is understandable given the flexibility of proline-induced kinks. Finally, a proteolytic fragment corresponding to residues 163-231, and therefore containing helices F (167-191) and G (203-225), has been studied in chloroform/methanol (Barsukov et al., 1992). Two helical regions were detected (168-191 and 198-227) which correspond well to TM helices in intact bR. Helix F exhibited a proline-induced kink of 25 ~ comparable to a kink angle of 30 ~ in

Principles of Membrane Protein Structure

49

intact bR. However, in this isotropic solvent, helices F and G did not pack together. This provides evidence that a bilayer environment is required to restrict the topology of packing of TM helices. Studies on bR and on lac permease have demonstrated that fragments containing one or more TM helices may insert into bilayers and pack with their partner fragments so as to generate functional proteins. For example, chymotryptic cleavage of bR between helices B and C yields two fragments which will refold in a bilayer to form functionally active bR, which in turn will form 2D crystals indistinguishable from those of the native protein (reviewed by Popot and Engelman, 1990). Furthermore, the five-helix (C to G) proteolytic fragment will refold with two synthetic peptides corresponding to helices A and B to form functional bR (Kahn and Engelman, 1992). Such results are not limited to bR. For example, a series of studies using pairs of polypeptide fragments of lac permease (from independently cloned fragments of the lac Y gene) reveal that comparable functional association within a bilayer can occur for this 12-helix IMP (reviewed by Kaback, 1992). For example, pairs of fragments corresponding to helices 1-6 and 7-12, to helices 1-2 and 3-12, orto helices 1 and 2-12 will associate within bilayers to form functional permease. Overall, these two sets of investigations provide compelling experimental evidence for the second stage of the two-stage folding model. Overview of Prediction

The results of topological prediction and analysis provide definition of TM helices and how they span the bilayer. Furthermore, if an interhelix loop is relatively short, then two TM helices adjacent in the sequence are likely to be adjacent in the 3D structure. The aim of structure-prediction studies is to take such information and by combining it with additional, experimentally-derived restraints, to generate models of TM helix packing in intact IMPs. What types of restraints may one place upon packing of TM helices within a bundle? If one considers a bundle of seven TM helices, as in bR, a reasonable restraint on possible packing models concerns the orientation of the helices within the bundle, i.e., which surfaces of the helices point towards the interior of the bundle, and which surfaces make contact with surrounding lipid molecules. Some progress has been made in deriving such restraints from sequence information. What information can one expect such predictive studies to generate? Certain aspects of TM helix packing may be answered by simulation studies. For example, do helices pack as a left-handed (as in bR; see Figure 2) or a right-handed bundle? How do helices pack relative to one another, i.e., what are interhelix contacts and helix-crossing angles? To what extent are TM helices tilted relative to the bilayer normal? Even if predictive studies cannot accurately place the side-chains of TM helices, one hopes that simulations of TM helix packing may answer such "lowresolution" structural questions.

50

M.S.P. SANSOM and IAN D. KERR

Overall, the input for simulations of TM helix packing should consist of idealized bundles, with the predicted interior side-chains pointing towards the center. Discrimination between alternative models resulting from simulations of packing relies on geometric and energetic analysis of models, and their comparison with experimental data. In the ensuing discussion, we explore how such methods have been applied to bR and to other all-t~ IMPs.

Helix Orientation In order to simulate packing of TM helices, it is necessary to predict an approximate orientation of the constituent helices within a bundle. That is, for each helix one must predict which residues are buffed within the interior of the bundle and which residues are exposed to the surrounding lipid environment. Two approaches to this problem are described, one based upon comparison of TM sequences from homologous proteins, and one based on analysis of TM helix amphipathicity.

Sequence Variability. Yeates et al. (1987) compared the sequences of TM helices of PS/RC proteins from three closely related species (Rb. sphaeroides, R. viridis, R. capsulata). The overall degree of sequence conservation was 50% for the L and M subunits, but this fell to only 16% for those residues deduced to be exposed to the surrounding lipid on the basis of the R. sphaeroides crystal structure. This resulted in the proposal that lipid-exposed residues of TM helices are poorly conserved, whereas those buried within helix bundles are more highly conserved. This is presumed to reflect the ability of a fluid bilayer to pack around a hydrophobic protein surface regardless of the exact shape of the surface. This initial observation has been extended by employing Fourier transform analysis to identify a surface of maximum variability (and hence a lipid-exposed surface) of TM helices. Komiya et al. (1988) used such analysis to detect periodicity of conserved/nonconserved residues for six species of PS/RC protein, providing a rigorous demonstration of the greater variability of lipid-exposed residues. Rees et al. (1989) extended this analysis to 82 sequences taken from six different families of all-0t IMPs. The relationship between the most variable surface and the most hydrophilic surface (defined by hydrophobic moment analysis) of 35 putative TM helices was examined. The mean angle between the variable and hydrophilic faces of the helices was 129 ~ and the mode of the corresponding angular distribution was ca. 180~ This suggests that interior residues of TM helix bundles are more hydrophilic than exposed residues. However, the mean hydrophobicity of interior residues of TM helices was equivalent to that of the interior residues of helices in hemoglobin. Thus, the interior of a TM helix bundle is of equal hydrophobicity te the interior of a globular protein, whereas the surface of a TM helix bundle is more hydrophobic than its interior, and exhibits considerable sequence variability. Extensions and elaborations of this basic approach have been described. Foi example, Donnelly et al. (1993) used Fourier analysis of sequence variability

Principles of Membrane Protein Structure

51

between 33 PS/RC sequences in order to calculate a substitution table for lipid-facing residues of TM helices. This was employed with some success to predict the orientation of helices within the bR helix bundle. Cronet et al. (1993) analyzed the sequences of bR and three related proteins by dividing each TM helix into eight angular zones, ranging from completely exposed to completely internal. This was used to generate a preference parameter (i.e., preferred angular zone within a TM helix in a bundle) for each of the 20 amino acid residues. Expansion of the sequence and structure databases for all-o~ IMPs will allow further refinement of these types of methods.

Helix Amphipathicity. As discussed above, e.g., Rees et al. (1989) have shown that, in general, the interior of a TM helix bundle is more hydrophilic (less hydrophobic) than the lipid-exposed outer surface. This is expected to be particularly so for IMPs, such as ion channels and transport proteins, in which the center of the bundle provides a relatively hydrophilic pathway whereby an ion may cross a membrane. It, therefore, is possible to predict TM helix orientation by defining the most hydrophilic surface. At the level of an amino acid sequence, this may be achieved by calculation of hydrophobic moments. When applied to bR (see e.g., Cronet et al., 1993) such analysis correctly predicts the orientation of helices A, C, and (to a lesser extent) G, fails completely for helices B, E, and F, and places the hydrophilic face of D at an interfacial location. An alternative approach to defining the hydrophilic surface of a TM helix has been developed by Kerr and Sansom (1993a) in the context of ion channels formed by bundles of amphipathic helical peptides. This method requires an atomic resolution model of a TM helix. This model is used to estimate the potential energy of interaction of a water molecule with the protein while placing the water molecule at successive points on a cylindrical grid centered on the helix. The helix-water interaction energies thus defined are displayed as a hydrophilic surface map (HSM). Calculation of HSMs for the (experimentally determined) TM helices of bR correctly predicted the orientation of helices C, E, and G, and placed the hydrophilic faces of A and D at helix interfaces (Kerr and Sansom, 1993b). The method failed for helices B and E However, helix B is not very amphipathic. When applied to helices of unknown three-dimensional structure, simulated annealing (see below) is used to generate an ensemble of models of a helix. The members of the ensemble differ in their side-chain conformations. The HSM may be calculated for each member of an ensemble, and so the degree of variation in the position of the center of the hydrophilic face may be estimated. An example of this method as applied to 8-toxin (a channel-forming peptide from Staphylococcus aureus) is shown in Figure 5. The sequence of 5-toxin is: fM-A-Q-D-I-I-S-T-I-G-D-L-V-K-W-I-I-D-T-V-N-K-F-T-K-K The alternation of hydrophobic (Roman) and hydrophilic (italic) residues within this sequence results in a helical structure (Tappin et al., 1988) of marked amphi-

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Principles of Membrane Protein Structure

53

pathicity. The HSM (Figure 5B) reveals a clearly defined hydrophilic surface generated by aspartate side-chains at positions 4, 11 and 18 of the helix. However, variation of side-chain conformations between members of the ensemble (Figure 5A) results in a 20 ~ range in the position of the center of the hydrophilic face. By suitable averaging of the HSMs (Figure 5C), a hydrophilic face may be defined which has the Co~ atom of residue D 11 at its center. This has been used to develop a preliminary model of ion channel formation by a bundle of six parallel 8-toxin helices which may subsequently be refined by simulated annealing (see below).

Simulation Studies In this section, we describe a number of attempts to simulate packing of TM helices within bundles. After reviewing early studies, and investigations of TM helix dimers, we focus on two areas which have been investigated in more detail: (a) parallel helix bundles of ion channels, and (b) bR and related seven-helix bundle proteins.

Preliminary Investigations. Pioneering studies of packing within TM helix bundles were carried out by Pullman and colleagues (Furois-Corbin and Pullman, 1986a,b; 1987a, b). Energy minimization methods were used to pack together Alan helices, treating the latter as rigid bodies. Initial studies examined the stability of Alan dimers and the contributions of different energy terms to dimer stabilization (Furois-Corbin and Pullman, 1986a,b). The electrostatic stabilization increased with increasing n until it reached a plateau at n = 14, whereas the van der Waals stabilization continued to increase with increasing helix length. The van der Waals interactions resulted in stabilization of both parallel and anti-parallel dimers (-17 a n d - 1 4 kcal mo1-1 respectively, for n = 14), despite the unfavorable electrostatic interactions in the former. For both dimers, the interaxial separation of the helices was ca. 8 *. A range of Ala14 helix bundles made up of N alternating antiparallel helices (PN for N = 3 to 7) were examined as possible models of transbilayer pores. The P3 bundle had no central pore, the P4 bundle had a narrow central pore, while the P5 bundle had a pronounced pore running throughout the length of the bundle. The P6 and P7 bundles, initially possessing 6x and 7x symmetry, were considerably distorted during energy minimization, the helix bundles "collapsing" inwards so as to prevent formation of a central pore. At a superficial level the "collapsed" P7 bundle resembled the seven-helix bundle of bR and related proteins. TM Helix Dimers. More recent investigations have employed more powerful simulation methods. In particular, simulated annealing by restrained molecular dynamics (SA/MD) has been used to explore possible packing of TM helix dimers. The SA/MD method is closely related to that used in the determination of globular protein structures on the basis of nuclear Overhauser effect (NOE) restraints derived from NMR experiments (Brtinger and Karplus, 1991; Nilges and Brtinger, 1991).

54

M.S.P. SANSOM and IAN D. KERR

It generates an ensemble of structures, all of which satisfy distance restraints chosen in order to embody biochemical data. Variation between members of an ensemble enables one to ascertain the extent to which a set of distance restraints specify a final structure. A major advantage of SA/MD is that it explores a wider range of conformational space than e.g., energy minimization, so the final structure is less biased towards the initial model. Kerr et al. (1994a,b) have used SA/MD to model parallel Leu20 helix dimers, chosen as simple models of hydrophobic TM helices. The initial model was a Cot atom template corresponding to an exactly parallel (f~ = 0 ~ pair of idealized t~-helices. All atoms other than the Coc atoms were added automatically during the SA/MD run. The resultant ensemble of structures (Figure 6A) had a mean crossing angle of f~ = +18 ~ corresponding to classical "ridges-in-grooves" packing of helices. Similar results were obtained for Ala20 helix dimers. Mean interaxial separations were R = 9.3 and 8.1 ~ for Leu20 and Ala20 helices, respectively. This study demonstrates that SA/MD will automatically pack hydrophobic helices into a favorable geometry with no explicit input other than an approximate Cct atom model and with only weak distance restraints in order to maintain the helices in an approximately parallel orientation. A similar SA/MD method was used by Treutlein et al. (1992) to model dimers formed by the TM helix of glycophorin. In vitro mutagenesis studies (Lemmon et al., 1992) revealed that dimerization of glycophorin TM helices in a detergent environment is sequence specific, and that subtle changes in sequence could alter helix-helix association. Both mutagenesis and SA/MD studies suggested that polar residues did not play an important role at the glycophorin helix-helix interface. Interestingly, the most stable packing of glycophorin TM helix dimers corresponded to f~ = -30 ~ This differs significantly from the classical "ridges-ingrooves" packing of Leu20 helix dimers (above), suggesting that specific side-chain-side-chain interactions must be taken into account when modeling

Figure 6. Models of bundles of TM helices generated by SA/MD. A shows 10 structures selected from an ensemble of 50 models for a parallel dimer of Leu20 helices. The oc-carbon backbones of the helices are shown superimposed. B illustrates a single structure from the ensemble in helical ribbon format. The helix axes, indicated by broken lines, define a helix crossing angle of D. = + 18 ~ C and D are ribbon diagrams of a parallel bundle of five Leu20 helices. In C the N-termini of the helices are uppermost and in D they are towards the viewer. The helix axes in this latter diagram indicate a positive crossing-angle between adjacent helices, resulting in a left-handed supercoil. E and F show a hexameric bundle of parallel helices used to model ion channels formed by Staphylococcus aureus G-toxin. In F, the side-chains of residues D4, D l l and D18 which line the central pore are shown in "ball-and-stick" format. (A to Dmi. D. Kerr et al., 1994a,b; E and Fml. D. Kerr, R. Sankararamkrishnan, and M. S. P. Sansom, in preparation).

:

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E

F 55

56

M.S.P. SANSOM and IAN D. KERR

packing of TM helices. These studies provide further evidence that modeling via SMMD can provide new information about TM helix packing beyond that included in the initial model. Parallel Helix Bundles and ion Channels. A parallel bundle of TM helices is a structural motif thought to be present in several ion channel proteins (Oiki et al., 1990). This motif is of particular importance with respect to the nicotinic acetylcholine receptor (nAChR) superfamily. As discussed below, the central pore of the nAChR is formed by a bundle of five parallel amphipathic helices (Unwin, 1989, 1993; Stroud et al., 1990; Sansom, 1992a, 1993c). A similar motif may occur in other ion channel proteins. For example, the influenza M2 channel can be modeled as a bundle of four parallel t~-helices (Sansom and Kerr, 1993). Helix bundles are also found in channels formed by a number of channel-forming peptides (CFPs) e.g., alamethicin (Sansom, 1993a, b) and S. aureus &toxin (Mellor et al., 1988; Sansom et al., 1991). Furthermore, synthetic peptides designed to form amphipathic ct-helices generate cation-selective channels when incorporated into lipid bilayers (Lear et al., 1988). It is, therefore, of some interest to simulate packing within bundles of parallel TM helices. SA/MD has been used to model parallel bundles of Ala20 and Leu20 helices (Kerr et al., 1994a). Interhelix distance restraints were applied to maintain approximate symmetry of the bundles, and thus to permit formation of a central pore. An example of a pentameric bundle of Leu20 helices is shown in Figure 6C. This is from an ensemble of 20 such structures for which the mean crossing angle was f~ - + 13 ~ A positive crossing-angle generates a bundle corresponding to a left-handed supercoil, as commonly found in coiled-coil structures (Parry and Cohen, 1990). Corresponding left-handed supercoils were formed by Ala20 helices, and by tetrameric and hexameric bundles. This suggests a left-handed supercoil is a favorable structure for parallel, symmetric bundles of hydrophobic TM helices. However, packing changed slightly once polar residues were incorporated into the TM helices. Introduction of serine and threonine residues resulted in smaller crossing angles (f~ ca. +5~ apparently in order to accommodate interhelix H-bonds stabilizing the bundles. This parallels the observation on glycophorin dimers that TM helix packing is sequence dependent. The same SA/MD methodology has been used to model ion channels formed by parallel bundles of CFP helices. For example, hexameric bundles of S. aureus &toxin (see above) helices have been modeled by Kerr et al. (I.D. Kerr, R. Sankararamakrishnan, and M.S.P. Sansom, in preparation; see Figure 6E, F). The initial Co~ template was constructed such that the Cot of residue D 11 was directed towards the center of the bundle. In light of the results with Ala20 and Leu20 bundles, the 8-toxin helices were tilted so as to generate an initial crossing angle of f~ - +20 ~ The mean crossing-angle for the resultant ensemble of eight structures was f~ = 19 (+ 2) o. The side-chains of D4, D11 and D18 line the central pore, creating a hydrophilic lining to the central pore. This provides a pathway whereby ions may

Principles of Membrane Protein Structure

57

cross a planar lipid bilayer. Polar side-chains also contribute to stabilization of the helix bundle by formation of interhelix H-bonds and electrostatic interactions. Overall, these studies demonstrate the potential of SA/MD for modeling ion channels formed by bundles of parallel TM helices. It is likely that the method will be further refined and applied to a wider range of channels and transport proteins.

Bacteriorhodopsin. There have been several attempts to simulate packing of TM helices within bR. For example, Jfihnig and Edholm (1992) used molecular dynamics (MD) simulations to evaluate the extent to which tertiary structure-predictions were dependent on prior assumptions concerning the arrangement of the TM helices. These simulations were of particular interest in that a simple hydrophobic potential was used to mimic the presence of a lipid bilayer. This hydrophobic potential had been developed in earlier MD simulations of interactions between the glycophorin TM helix and a lipid bilayer (Edholm and J~nig, 1988). Starting with the helices of bR arranged on the circumference of a circle, it was found that although 25 ps MD simulations resulted in more compact helix bundles, the characteristic seven helix bundle ofbR was not generated. If the helices were placed at their approximate positions in the bR bundle, but with all helices exactly parallel to the bilayer normal, MD simulations then resulted in the TM helices adopting the same tilt angles relative to the bilayer as found in bR. Thus, the helix crossing-angles in the model must have been close to those in bR. Chou et al. (1992) adopted a different approach to modeling bR. The initial model was based on fitting seven TM helices to the experimental bR structure and then refining the resultant model using a combination of simulated annealing and energy minimization. The calculations were performed twicemonce with all-trans-retinal (as in the experimental bR structure) and once with 13-cis-retinal (as in the light-activated state). An energetic analysis of the resultant models was performed. It was concluded that while electrostatic interactions favored the antiparallel packing of adjacent helices, van der Waals interactions drove most adjacent helices close to the ideal packing angle of f~ = -160 for "ridges-in-grooves" packing of antiparallel helices. Indeed, examination of published helix-crossing angles (Table 6) reveals this is the case for these models of bR and for a model of a seven-helix G-protein-coupled receptor (see below). Together these and related studies encourage the use of simulations of TM helix packing to model all-(z IMPs. We will now briefly examine some modeling studies of seven-helix IMPs other than bR. G-Protein-Coupled Receptors. These proteins constitute a superfamily of IMPs whose amino acid sequences contain seven hydrophobic segments. They include rhodopsin, the recent 2D structure determination of which has provided experimental evidence for the presence of seven TM helices. On the basis of analysis of sequence variability within the superfamily, and of constraints imposed by connectivity between adjacent helices, Baldwin (1993) allocated the seven

58

M.S.P. SANSOM and IAN D. KERR

Table 6. Helix Crossing-Angles in Model 7-Helix Bundles fl(~ Helix Pair

bR E x ~

bR TRANs

bR cIs

~12AdR

A-B

-155

-150

-154

--170

B-C

-167

-170

-169

-163

C-D

+173

+168

-170

-170

D-E

-160

-151

-167

-174

E-F

-150

-169

-170

-178

F-G G-A

-150 +8

-165 +10

-162 +16

-171 +6

Notes: abREXrr--experimentally determined bR structure (Henderson et al., 1990); bRTRANS---modelof bR helix bundle containing all-trans retinal, and bR~ of bR helix bundle containing 13-cis retinal (Chou et al., 1992); l~2AdRumodel of 132-adrenergicreceptor (MaloneyHuss and Lybrand, 1992).

hydrophobic segments in the sequence to the seven peaks in the 2D projection map of rhodopsin. As in bR, the seven helices are packed in a left-handed fashion, with helices A and G parallel to one another and "closing" the seven-helix bundle. There have been several attempts to model G-protein-coupled receptors and their interactions with ligands. Carried out in the absence of a rhodopsin structure, most of these attempts have taken bR as their starting model. While such studies are of considerable interest as simulations of TM helix packing, results concerning receptor-ligand interactions should not be overinterpreted. Most of the model structures are quite close in their overall architecture to the initial bR structure. This is perhaps not surprising given that energy minimization was the principal methodology employed in these studies. For example, MahoneyHuss and Lybrand (1992) modeled a l~2-adrenergic receptor by a combination of interactive modeling and energy minimization. Helix packing within the resultant model is an approximate mirror image of that in bR. Homology modeling was adopted by Livingstone et al. (1992) to model dopamine receptors. Amore ambitious approach was adopted by Sylte et al. (1993), modeling a 5-HTla (serotonin) receptor. Sequence alignment and hydrophobicity profile analysis were used to define the seven TM helices. Seven isolated helices were generated and energy minimized so as to regularize side-chain conformations. These helices were interactively packed into a left-handed bundle with their most polar surfaces directed towards the center of the bundle. Interhelix loops were added, and the resultant model energy minimized, followed by a 25 ps MD simulation. Serotonin was added at the putative binding site and a further 100 ps MD simulation performed. The resultant structures clearly differed from that of bR. Unfortunately, from the published information it is not possible to assess to what extent it may match the projection structure of rhodopsin.

Proline in TM Helices. An important aspect of TM helix conformation which is particularly amenable to analysis via simulation is that of the structural role of

Principles of Membrane Protein Structure

59

intrahelical proline residues. Intrahelical prolines occur with a relatively high frequency in IMPs. For example, Brandl and Deber (1986) conducted a statistical survey of putative TM helices of IMPs and concluded that most transport proteins contain intramembranous prolines, whereas this residue is absent from TM helices of membrane proteins which lack a transport or channel function. They proposed two possible roles for intrahelical prolines: (a) facilitation of conformational changes via cis-trans isomerization of Xaa-Pro peptide bonds, and/or (b) provision of cation-liganding sites by the carbonyl oxygen of the Xaa-Pro peptide bond. In their review of ion channel structure, Eisenman and Dani (1987) highlighted a further possible role, stating that an intrahelical proline "frees a non-H-bonded backbone carbonyl oxygen for liganding to a cation". This was further explored in modeling studies of possible cation binding sites introduced into helix bundles by proline residues (Sansom, 1992b). The proline-kinked helices of bR and of PS/RC were analyzed by von Heijne (1991) who concluded that the convex face of a proline-kinked helix is often directed towards the center of a TM helix bundle. Woolfson et al. (1991) analyzed proline residues in amphipathic TM helices from a range of channel and transport proteins, revealing that proline is preferentially located on the hydrophilic face of such helices. On this basis they suggested that proline-kinked helices may form funnel-shaped pores, similar to those proposed as the basis of ion channel formation by alamethicin (Fox and Richards, 1982; Sansom, 1993a,b)and related CFPs (Sansom, 1991; Balaram et al., 1992). Although little is known of their structural role in IMPs other than bR and PS/RC, possible structures may be inferred by analysis of intrahelical prolines in globular proteins. B arlow and Thornton (1988) and Sankararamakrishnan and Vishveshwara (1992) have analyzed kink angles ofproline-containing helices in globular proteins. Both studies arrived at a mean kink angle of 25 ~ with kink angles varying from 9 ~ to 49 ~ Such crystallographic data suggest that proline-induced kinks may produce flexible "hinge" regions between two rigid segments within a TM helix. A number of theoretical studies have addressed this possibility. Polinsky et al. (1992) used a molecular mechanics approach to search for stable conformations of an A8LPFA8 helix. Three low energy trans-proline conformations were described, with kink angles of 29 ~ 78 ~ and 83 ~ It was suggested that interconversions between these might permit hinge-bending movements. A similar conclusion was drawn by Yun et al. (1992) using MD simulations to estimate the free energy change on reducing the kink of a Ac-AsPAs-NHMe helix from 40 ~ to 15 ~ A value of < + 0.5 kcal/mol was obtained, suggesting that hinge-bending is thermodynamically feasible. Hingebending of Ac-A7WA2YPA2WAs-NHMe (a simplified model of helix F of bR) was investigated in MD simulations by Sankararamakrishnan and Vishveshwara (1993). The kink angle varied between 0 ~ and 50 ~ on a 10 ps timescale. A subsequent comparative study of Ac-AI2TPA10-NHMe, Ac-A13PATA8-NHMe, and Ac-A13PA3TA6-NHMe, (Vishveshwara and Vishveshwara, 1993) showed that varying the position of threonine relative to proline modulated the extent and

60

M.S.P. SANSOM and IAN D. KERR

dynamics of variations in kink angle. Simulations of the dynamics of Dns(AUAUA)3PAUAUAW-OMe, an analogue of a synthetic channel-forming peptide, yielded variations in kink angle of between 5 ~ and 45 ~ (Vogel, 1992). Overall, it is highly likely that proline residues within TM helices provide molecular "hinges". Such flexible structural elements may play key roles in the function of transport and receptor IMPs. For example, a strictly conserved proline residue in the second TM helix of connexin seems to play a key role in voltage-gating of gap junction channels (Suchyna et al., 1993).

Overview of Simulation Studies. Molecular modeling studies of all-@ IMPs are at an early stage of development. Further work is required before one may anticipate accurate simulations of TM-helix packing. In particular, developments are needed in: (a) methods for incorporating "biochemical" information in MD simulations (e.g., via the use of distance restraints or additional potential energy terms), (b) use of multiple and/or long MD runs in order to minimize bias towards the starting model, and (c) application to a wider range of all-~ IMPs in order to evaluate the extent to which methods are limited to particular families of all-o~ IMPs. Assuming that these problems can be solved, it is likely that simulation studies will enable accurate prediction of helix packing within all-@ IMPs.

IV. ALL-I~ IMPs This section is concerned with those IMPs which exhibit an all-13 topology. Experimentally determined structures exhibiting this topology will be described, followed by a discussion of other IMPs believed to fall into this class, and by a brief examination of attempts at structure-prediction for all-~ IMPs.

A. Experimentally Determined Structures Por/ns The structures of four prokaryotic porins have been determined at high-resolution (Table 1; reviewed by: Schirmer and Rosenbusch, 1991; Cowan, 1993). All have the same basic fold, first observed in Rb. capsulatus porin, and so the latter structure will be described in detail. Porins are found in outer membrane of Gram-negative bacteria. They form water-filled transbilayer pores, of diameter ca. 10/~, which allow uptake and loss of small hydrophilic compounds through this membrane. As such they act as "molecular sieves" with an exclusion size of ca. 600 Da. The pores allow passive diffusion across the bilayer, are weakly ion selective, and exhibit some degree of voltage-gating. The structure of Rb. capsulatus porin (Weiss et al., 1991) is shown in Figure 7. The functional molecule is a trimer, each monomer of which forms a transbilayer

A

B Figure 7. Rb. capsulatus porin (coordinates from entry 2POR of the PDB). [3-Strands are represented by arrows. A is a porin monomer, viewed parallel to the plane of the membrane with the face of the barrel closest to the three-fold axis towards the viewer. B a porin trimer, viewed perpendicular to the membrane plane (i.e., down the three-fold axis). The three parallel pores are clearly visible. The three dark spheres represent interfacial Ca 2+ ions.

61

62

M.S.P. SANSOM and IAN D. KERR Table 7. Porins: TM 13-Strands and Loops

Poffn Rhodobacter capsulatus Rhodopseudomonas blastica E. coli (OmpF)

~-Strand Length ExternalLoop PeriplasmicLoop (Mean + SD) Length(Mean + SD) Length (Mean + SD)

10.7 + 2.9 10.8 + 2.5 12.4 + 2.6

13.8 + 12.6 12.6 + 12.9 15.4 + 7.5

2.3 + 0.5 2.3 + 1.7 2.4 + 2.2

pore. The monomer is a 16-strand antiparallel 13-barrel. Of the 301 residues, [~-strands account for 57% of the amino acid residues. The [3-strands are tilted relative to the trimer axis by between 30 and 60 ~ and the barrel has a right-handed twist. 13-strand lengths range from 6 to 17, with a mean length of 11 residues (Table 7). The height of the barrel wall correspondingly varies from 20 to 40 ]k, with the lowest region of the wall at the trimer interface. Interactions at the trimer interface are primarily polar, as are the residues which form the lining of the pore. The equatorial surface of the trimer is made up of hydrophobic side-chains. Thus the 13-strands in this region are amphipathic, with alternating hydrophobic (external) and hydrophilic (internal) side-chains. Interconnecting loops are short (two or three residues) at the periplasmic end of the barrel, but are more extensive at the external end. An extended external loop folds back into the barrel to form a so-called "eyelet" which is responsible for the size exclusion limit of the pore. The R. blastica (Kreusch et al., 1994) and E. coli (Cowan et al., 1992) porins exhibit a similar architecture to that just described (Table 7). I]-strand and loop lengths for the R. blastica and Rb. capsulatus porins are very similar. The E. coli porins both have 16-strand 13-barrels, but exhibit subtle differences from the other two porins. In both OmpF and PhoE the topology is pseudocyclic in that the 16th strand is made up of both the N- and C-terminal strands, with an intrastrand salt-bridge between the polypeptide chain termini. On average, the 13-strand length is somewhat higher for the E. coli porins, as is the external loop length (Table 7). An interesting feature of all of the porin structures is the distribution of aromatic residues on the outside of the [~-barrel. These form two rings around the trimet surface, one each side of the central hydrophobic band. This is reminiscent of the distribution of tryptophan residues in the PS/RC structure (see above and Schiffer et al., 1992). It has been suggested that in porins, such aromatic residues may play a role in "shielding" the protein molecule against adverse membrane fluctuations (Kreusch et al., 1994). An alternative explanation is that aromatic side-chains, particularly tyrosine and tryptophan, interact favorably with polar lipid headgroups in the bilayer/water interfacial region. Full evaluation of the quantitative importance of such interactions will require detailed structural information on thei~ molecular nature. The porin structures provide some clues as to the latter. Fo~ example, in the R. blastica crystal structure, electron density is present at the trime~ interface corresponding to the n-alkyl chains of bound detergent molecules.

Principles of Membrane Protein Structure

63

A second unusual feature of porins is the distribution of charged side-chains. There is a marked segregation of charge within the pore, with a cluster of positively charged side-chains close to the trimer axis and of cluster of negatively charged side-chains on the opposite face of the pore lining. This creates a strong transverse electrostatic field across the pore, which has been suggested to play a role in governing the selectivity of porin channels (Weiss et al., 1991a).

Aerolysin It is perhaps premature to classify aerolysin as an "experimentally determined structure". However, a wealth of experimental data points towards a predominantly I3-sheet structure for this protein. Aerolysin is a channel-forming toxin produced by Aeromonas hydrophila, a Gram-negative bacterium. It is secreted as proaerolysin, a 54-kDa soluble protein, which is converted to aerolysin by proteolytic removal of a C-terminal peptide of ca. 40 residues. Sequence analysis of proaerolysin fails to reveal any TM helices. It is, therefore, of interest to determine how a hydrophilic globular protein is converted to a channel-forming IME Proaerolysin has been shown spectroscopically to contain predominantly I3-sheet (Buckley, 1992). The crystal structure has been solved at 2.8/k resolution, and has been shown to consist of 40% I3-sheet (Tucker et al., 1990; Parker et al., 1994). The transbilayer pore formed by aerolysin has been imaged at 25/~ resolution by EM of 2D crystals (Wilmsen et al., 1992). The pore is formed by a heptamer of aerolysin molecules, packed in "barrel-stave" fashion about a central pore, diameter 17 A, the pore axis being coincident with the sevenfold symmetry axis. By combining the EM and X-ray derived structures, a model was generated in which the pore is lined by 21 I3-strands (Parker et al., 1994). A series of elegant experiments have provided valuable clues concerning the pathway from proaerolysin to aerolysin pores (van der Goot et al., 1993). Proaerolysin binds to cell membranes and undergoes proteolysis and subsequent oligomerization before inserting into the bilayer to form channels. Changes in binding of the hydrophobic dye, 8-anilino-l-naphthalene sulfonate (ANS), suggest that oligomerization exposes hydrophobic surface patches which drive membrane insertion. Therefore, there is a conformational transition from globular protein to IME However, in view of the convergence of the EM and X-ray results described above, it is clear that the latter retains a predominantly 13structure.

B. Related Structures

Porin-Like Proteins How widespread are all-l~ IMPs? It seems that some other proteins of bacterial outer membranes may possess porin-like folds. For example, high-affinity, ligandspecific transport proteins may be composed of a "gated-porin" along with a

64

M.S.P. SANSOM and IAN D. KERR

"gate-keeper" protein which facilitates entry of ligands into the porin-like channel (Rutz et al., 1993). Gated-porin proteins are predicted to have an antiparallel l-barrel structure, with the external loops responsible for binding of specific ligands. In eukaryotes, porins are present in outer mitochondrial membranes, where they are also referred to as VDACs (voltage-dependent anion channels; reviewed by Mannella, 1992). A low resolution projection-structure from EM studies of Neurospora crassa mitochondrial porin reveals cylindrical pores of ca. 30 ,~ diameter. These have been modeled as consisting ofbetween 12 and 19 amphipathic [3-strands in a barrel-like structure. It will be interesting to determine the extent to which the structures of mitochondrial porins resemble those of prokaryotic porins. Overall, there is little evidence suggesting that all-13 topologies are as widespread among IMPs as are all-~ topologies. However, it may be that further examples await discovery. For example, Fischbarg et al. (1993) have described a somewhat speculative porin-like model for mammalian facilitative glucose transporters.

TopologyPrediction There are two elements to structure-prediction as applied to putative porin-like topologies (Jahnig, 1989). First, it is supposed that the pore-lining ]3-strands will be amphipathic, with hydrophobic external side-chains and hydrophilic internal side-chains. Such a secondary structure is characterized by a very simple sequence patternmalternating hydrophobic and hydrophilic amino acids. Generally amphipathic 13-strands of 9 or 10 residues in length are searched for, in agreement with the length of such strands in experimentally determined porin structures. The second element is to predict the positions ofinterstrand l-turns. This may be carried out using a standard secondary structure-prediction method, such as that of Chou and Fasman (1978). An early attempt at topology prediction for porin, maltoporin, and OmpA protein from E. coli was made by Vogel and Jahnig (1986), in parallel with Raman spectroscopic measurements to determine the secondary structure composition of these proteins. The two porins were predicted to contain 18 amphipathic [3-strands per monomer, forming a 13-barrel around a central pore. The OmpA 1-177 fragment was predicted to form an eight-strand ]3-barrel. It has since been demonstrated that OmpA forms pores in lipid bilayers, with a low (relative to porins) pore diameter (Saint et al., 1993). A more recent predictive study on members of the porin superfamily was carried out by Jeanteur et al. (1991). Alignment of 14 porin sequences was combined with secondary structure-prediction of amphipathic 13-strands and [3-turns. The result was a consensus prediction of 16 13-strands, thus correctly predicting the number of TM ]3-strands in E. coli OmpF and PhoE porins, the structures of which were not published at the time of this work.

Principles of Membrane Protein Structure

65

Overview Topology predictions appear to work quite well for IMPs which exhibit a porin-like fold. It remains to be seen how widespread this class of fold is within other families of membrane proteins. It is possible that it is a rather specialized structure adopted when a large diameter, weakly selective transbilayer pore is required.

VI. ~/p IMPs In this section we discuss two families of ion channel proteins believed to have a "mixed" TM topology, containing both (z-helices and [3-strands. In neither case is a high resolution structure available, but in both cases evidence in favor of an (z/13 topology is reasonably convincing. In addition to reviewing these two families of IMPs, the possible general relevance of such topologies will be discussed.

A. Nicotinic Acetylcholine Receptor Structure The nicotinic acetylcholine receptor (nAChR) is the most intensively studied member of a superfamily of ligand-gated ion channels. The nAChR is a cation-selective channel found in post-synaptic membranes, which may be obtained in large amounts from Torpedo(an electric fish). It has been investigated using a wide range of biophysical techniques, from cryo-electron microscopy to patch-clamp recording, and also subjected to functional dissection by chemical labeling and sitedirected mutagenesis. The structure determination at 9 A resolution of the nAChR from Torpedo (Unwin, 1993) provides a three-dimensional model of the receptor-channel. Previous structural studies at 17 ,~ resolution (Toyoshima and Unwin, 1988) defined the overall shape of the protein, and revealed that the five homologous subunits ((zz]3yS) are packed pseudosymmetrically about a central axis which runs perpendicular to the plane of the bilayer. The ion channel appeared to lie along the fivefold axis. Hydrophobicity profile analysis of 50 or so nAChR sequences suggested that there were four TM helices (M1, M2, M3, and M4) per subunit. Circular dichroism experiments (Mielke and Wallace, 1988) introduced an element of doubt, as they demonstrated that the total (z-helical content of the nAChR was only 23%. If one assumed there to be four TM helices per subunit, this excluded the presence of (z-helices from elsewhere in the molecule, including the large extramembranous domain. However, the apparent conservation of the four TM helices in the sequences of other ligand-gated ion channels was persuasive evidence in favor of this model (Unwin, 1989; Betz, 1990). Mutagenesis and chemical

66

M.S.P. SANSOM and IAN D. KERR

labeling studies (Changeux et al., 1992) focused attention on the M2 helix, which was shown to interact with permeant ions and with channel-blocking drugs, and thus was proposed to line the ion channel. Determination of the structure of the nAChR used tubular crystals which presented the nAChR in a number of different projections, allowing direct reconstruction of the 3D structure of the protein from 2D images. At 9 A resolution, the most prominent elements of the secondary structure, the t~-helices, were evident. Overall, the nAChR is a 120 ,~ long, 80 ,~ diameter cylinder, with a pseudo-5x symmetry axis running down the center of the molecule perpendicular to the plane of the membrane. The ion channel lies on the fivefold axis. There is a 60/k long and 20 ,~ wide entrance to the channel on the synaptic face, followed by a narrow pore across the bilayer (about 30 ,~ long) which widens to form the cytoplasmic entrance, 20 A wide and 30 ,~, long. The 9 /k resolution map also revealed phospholipid headgroups and so allowed the protein to be positioned accurately relative to the bilayer. The synaptic (extramembranous) domain of the nAChR accounts for 55% of the mass of the protein. It includes, on the two t~-subunits, binding sites for the neurotransmitter acetylcholine. The synaptic domain of each subunit contains a left-handed coil of three t~-helices. In the t~-subunits, these three helices form a cavity near the center of the subunit, which is reached from the surface of the molecule by a deep cleft. This cavity is presumed to correspond to the acetylcholine binding site. The intramembranous domain of the nAChR exhibits a high degree of rotational symmetry. It contains only o n e TM helix per subunit. The central pore is lined by five such helices, but these are surrounded by a continuous rim of density which is presumed to correspond to a 13-barrel (see Figure 8A). The transmembrane helices visible in the nAChR are interpreted to be the M2 helices. The M2 helices are markedly kinked (Figure 8B), and are oriented such that at their closest approach they occlude the central pore, presenting a barrier to ion permeation. Thus, the structure is believed to correspond to the closed conformation of the channel. The kink angle, as measured from the data shown in Figure 8B, is ca. 44 ~ This is of comparable magnitude to the kinks in proline-containing helices, and yet proline is absent from the M2 sequence. The N-terminal segments of the helices form a left-handed supercoil (f~ ca. +17~ as observed in simple models of parallel TM helix bundles (see above). Tentative alignment of the M2 sequence to the helices suggests that the constriction in the channel, corresponding to the apex of the kink, is made up of a ring of conserved leucine residues. This has resulted in the proposal that these leucines form a "gate" which closes the channel to passage of ions. Revah et al. (1992) have shown that mutation of the corresponding Leu in a neuronal nAChR to a smaller residue results in changes in gating and conductance properties of the channel. On this basis, one may begin to speculate about the nature of channel opening. An attractive model of channel opening is one in the which helix orientation changes, and

oc-helix I~-sheet

A

B Figure 8. Schematic diagrams of the TM structure of the nicotinic acetylcholine receptor. A is a section through the intramembranous region of the molecule, viewed perpendicular to the bilayer plane. Five M2 helices, one donated by each subunit, line the central pore, and are surrounded by a band of (putative) [3-sheet. B illustrates the approximate structure of the pentameric bundle of M2 helices, viewed from the synaptic end (i.e., C-termini) of the helices. (Drawn from data in Unwin, 1993).

67

68

M.S.P. SANSOM and IAN D. KERR

thus opening the channel (Unwin, 1995). The surrounding 13-sheet may provide a scaffolding supporting such helix movements.

Verotoxin A possible model for the transbilayer domain of the nAChR is provided by the B subunits of two bacterial A-B toxins (heat-labile enterotoxin, Sixma et al., 1991; and verotoxin-1, Stein et al., 1992) whose crystal structures reveal them to be made up of a central bundle of five parallel {x-helices surrounded by a framework of [3-sheet (see Figure 9). Interactions between 13-strands from adjacent monomers hold the pentameric complex together, and there is a central pore passing through the molecule. Interestingly, it has been suggested that the B subunit of verotoxin-1 may undergo a conformational change and bring about translocation of the A subunit across the membrane of the target cell (Read and Stein, 1993). This further strengthens the proposal that such toxins may be used as models of nAChR-like od~ IMPs.

Figure 9. The structure of the B subunit pentamer of verotoxin (PDB entry 1 BOV), viewed down the approximate five-fold axis. The central pore is lined by five (z-helices, one from each subunit, surrounded by an outer rim of l-sheet.

Principles of Membrane Protein Structure

69

General Significance What is the overall importance of the nAChR structure? It clearly is of relevance to the other members of the ligand-gated channel superfamily, which are presumed to share a similar TM architecture. In the context of IMPs in general, the novel transbilayer architecture warns against uncritical use of hydrophobicity plots to generate models of transmembrane topology. Clearly one should pay close attention to spectroscopic data, particularly for those IMPs believed to contain extensive extramembranous domains. Although hydrophobic helices may be detected by sequence analysis, this does not demonstrate that they occupy TM locations. The need for experimental topology information (see above) is clearly emphasized.

B. Voltage-Gated Ion Channels This superfamily of IMPs includes voltage-gated K +, Na + and Ca 2+ channels (Hille, 1992). Of these, the molecular structure of voltage-gated K + channels is perhaps the best understood. However, in the absence of high resolution data, our understanding of K + channel structure is derived from sequence comparisons and analysis, and from site-directed mutagenesis studies. The latter have resulted in an interesting proposal concerning the topology of the transmembrane domain, in particular the pore region, and it is this which we will describe in more detail. Voltage-gated K + channels, perhaps the best characterized of which is the Shaker channel of Drosophila, are thought to form tetramers in which the four subunits are packed around a central pore (MacKinnon, 1991). Low resolution EM images of purified Shaker channel protein (Li et al., 1994), reveal a square-shaped complex, of dimensions ca. 80 x 80/k, with a large central hole. Sequence analysis of voltage-gated K § channels reveals six putative TM helices (Figure 10A). This pattern is conserved in the corresponding membrane domains of voltage-gated Na + and Ca 2+channels. Na + and Ca 2+channels contain four repeats of a domain corresponding to a single K + channel subunit within one polypeptide chain. Initial proposals for voltage-gated channel structure had a central pore lined by a bundle of amphipathic TM helices. However, recent mutagenesis data are incompatible with such a model and suggest a more complex topology (reviewed by Miller, 1991; Pongs, 1993). Briefly, mutagenesis results indicate that the loop (H5) between TM helices $5 and $6 controls the ion selectivity of K + channels, and also contains those residues responsible for binding toxins (e.g., charybdotoxin, CTX) and simple organic molecules (e.g., tetraethylammonium, TEA) which have been demonstrated to block the channel when it is open. Thus, H5 is concluded to form at least part of the lining of the channel. Furthermore, mutations at either end of the H5 sequence alter interactions with externally applied CTX or TEA, whereas mutations in the center of H5 alter interactions with internally applied TEA. The simplest interpretation of these results is that H5 forms a ]I-loop which traverses the bilayer and forms the lining of the pore of Shaker. In conjunction with evidence

70

M. S. P. SANSOM and IAN D. KERR Shaker $1 $ 2 $ 3 $ 4 $ 5 H5

IRK1 $ 5 H5 $ 6

$6

pore

A

B

,.S5

12 Figure 10. Model structures for K+ channel proteins. A and B show proposed topologies of single subunits of Shakerand IRK1 channels. The pore lining region (I-t5) is modeled as a 13-hairpin flanked by s-helices. Both channel proteins are tetramers, with the central pore formed by an eight stranded antiparalle113-barrel, as indicated in 12, a schematic view of such a channel perpendicular to the plane of the bilayer.

on the tetrameric structure of Shaker this has resulted in the proposal illustrated in Figure 10C, in which the central pore is formed by an eight-stranded antiparallel ~-barrel. Two strands are donated by each subunit, and the 13-barrel is in turn surrounded by a 6 • 4 = 24 TM helix bundle. There have been several attempts to model Shaker channels. Durrell and Guy (1992) used an interactive approach, combined with energy minimization, to model the entire Shakerprotein. Their model was used as the basis of informed speculation concerning possible mechanisms of voltage-dependent activation and inactivation of Shaker. Bogusz et al. (1992) focused on the central 13-barrel, which was modeled using energy minimization on the basis of a theoretical model of an optimal eight stranded 13-barrel structure. Their model was used for calculations of ion permeation energies. Such models demonstrate the feasibility of a "mixed" ~13 structure for K § channels. However, it should be remembered that there is no structural evidence for a [~-barrel. Indeed, synthetic H5 peptide in isolation forms tx-helices in a

Principles of Membrane Protein Structure

71

membrane environment (Haris et al., 1994), although this does not preclude adoption of a I]-strand conformation in the intact protein. What is the general relevance of the proposed structure for voltage-gated channels? A related fold has been suggested for a second family of K § channels, exemplified by an inward rectifier K + channel (IRK1; Kubo et al., 1993). Sequence analysis of IRK1 suggests a smaller TM domain, containing only two putative TM helices per subunit. The loop between the two helices shows sequence homologies with H5 from Shaker. The TM topology proposed for IRK1 is shown in Figure 10B, with two TM helices flanking a central H5 [3-loop. The general significance of Shaker-like topologies, outside ion channel proteins, is as yet unknown. However, it seems possible that further mutagenesis and structural studies may reveal 13-loops within transport proteins previously suggested to be all-t~ IMPs.

VI.

CONCLUSIONS

The past decade has witnessed considerable progress in unravelling the principles of membrane protein structure. The all-o~ family of IMPs is represented by several experimental structures, and more structures will be solved at high resolution in the near future. Furthermore, there is now a good understanding of how TM helices assemble within lipid bilayers. This, in turn, is enabling development of computational procedures to simulate such self-assembly, thus allowing prediction of all-or IMP structures. Rather less is known concerning other classes of IMP. The porins remain the major representative of all-[~ IMPs. It remains to be seen how generally applicable structural principles governing the porins are to other membrane proteins. As far as more complex transbilayer topologies are concerned, the nicotinic receptor provides a tantalizing glimpse of possible ot/13 folds within bilayers. Again, the general relevance of such structures remains uncertain. What might the future hold? Further IMP structures will be determined at high resolution, both by EM and by X-ray diffraction. Both methods are now mainly limited by problems of how to express sufficient material in order to enable growth of suitably diffracting 2D or 3D crystals (Schertler, 1992). In addition to such direct methods, a "hybrid" approach to membrane protein structure may be possible. Such an approach might combine: (a) definition of TM topology, (b) determination of key residues by site-directed mutagenesis, (c) use of solid-state NMR techniques to determine selected inter-residue distances (Smith and Peersen, 1992; Smith, 1993), and (d) computer simulation to integrate all of the experimental information into possible molecular models. Of course, the success of such an approach will depend on increased understanding of structural principles as derived from further experimental structures. An increase in the number of experimental structures will result in a refinement of ideas concerning general principles of membrane protein

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structure. This, in turn, will e n a b l e g r e a t e r u s e to b e m a d e o f the e x p a n d i n g m e m b r a n e p r o t e i n s e q u e n c e database.

ACKNOWLEDGMENTS This work was supported by the Wellcome Trust. Our thanks to our colleague Dr. R. Sankararamakrishnan for help with calculations on TM helix geometry.

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