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DTD 5 ARTICLE IN PRESS doi:10.1016/j.jmb.2004.11.036 J. Mol. Biol. (2004) xx, 1–9 A Study of the Membrane–Water Interface Region of Membrane Proteins Erik Granseth, Gunnar von Heijne and Arne Elofsson* Stockholm Bioinformatics Center, AlbaNova, SE-106 91 Stockholm, Sweden The most conspicuous structural characteristic of the a-helical membrane proteins is their long transmembrane a-helices. However, other structural elements, as yet largely ignored in statistical studies of membrane protein structure, are found in those parts of the protein that are located in the membrane–water interface region. Here, we show that this region is enriched in irregular structure and in interfacial helices running roughly parallel with the membrane surface, while b-strands are extremely rare. The average amino acid composition is different between the interfacial helices, the parts of the transmembrane helices located in the interface region, and the irregular structures. In this region, hydrophobic and aromatic residues tend to point toward the membrane and charged/polar residues tend to point away from the membrane. The interface region thus imposes different constraints on protein structure than do the central hydrocarbon core of the membrane and the surrounding aqueous phase. q 2004 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: membrane protein; protein structure; bioinformatics; interface helix Introduction a-Helical membrane proteins account for approximately 20% of all proteins in a typical genome.1 Over the past few years, several studies have been performed on the structural characteristics of a-helical membrane proteins. These studies have mainly focused on the transmembrane a-helices, describing their amino acid composition and packing interactions in considerable detail.2–6 In contrast, the parts of the proteins located in the membrane–water interface region have received little attention, and we are not aware of any detailed studies of the structural constraints imposed on proteins by this region of the lipid bilayer. Here, we present a first analysis of this interface region in a non-redundant set of a-helical membrane proteins of known structure. Our data set consists of 56 chains from 27 PDB files, Table 1. No one chain is more than 30% identical in sequence to any other chain in the data set. In total, the data set contains 221 transmembrane helices and 78 interfacial helices (see Materials and Methods for a precise definition of Abbreviations used: TM; transmembrane; PDB; Protein Data Bank. E-mail address of the corresponding author: [email protected] the different types of secondary structure). For comparison, we also include five peripheral membrane proteins in the study. The global view In agreement with earlier findings,2 the secondary structure composition in the G10 Å central region of the membrane is almost 100% helix, Figure 1(a). The helix content steadily decreases from the center of the membrane to a minimum at around G35 Å. The small amount of irregular structure in the central region originates from ion channels and aquaporin-like proteins that contain so-called re-entrant loops that extend part of the way across the membrane. Outside the central G10 Å region, the amount of irregular structure increases to a peak around G25 Å, and then decreases again. Appreciable amounts of b-strucstructure are found only G35 Å or more from the center of the bilayer. The distribution of different categories of amino acid residues is shown in Figure 1(b). As expected, the central region contains mainly aliphatic residues, but also a significant proportion of polar residues (30%). Polar residues, Gly and Pro in particular, are most frequent in the G20–30 Å region where most of the transmembrane helices end. Another noticeable feature is a peak of Trp and 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. ARTICLE IN PRESS DTD 5 2 Membrane–Water Interface of Membrane Proteins Table 1. Description of proteins used in this study, chains in bold contain interface helices, chains in italic contain reentrant loops TM helix protein PDB code Organism Chains Cytochrome c oxidase Cytochrome bc1 Potassium channel Bacteriorhodopsin Halorhodopsin ba3 cytochrome c oxidase Calcium ATPase Rhodopsin Ubiquinol oxidase Aquaporin Glycerol-conducting channel Sensory rhodopsin Multidrug efflux transporter Photosystem I Formate dehydrogenase Light-harvesting protein Fumarate reductase ABC transporter Light-harvesting complex Mechanosensitive channel Mechanosensitive channel Cytochrome c oxidase 1AR1 1BGY 1BL8 1C3W 1E12 1EHK 1EUL 1F88 1FFT 1FQY 1FX8 1H68 1IWG 1JB0 1KQF 1KZU 1L0V 1L7V 1LGH 1MSL 1MXM 1OCC Paracoccus denitrificans Bos taurus Streptomyces lividans Halobacterium salinarum Halobacterium salinarum Thermus thermophilus Oryctolagus cuniculus Bos taurus Escherichia coli Homo sapiens Escherichia coli Natronomas pharaonis Escherichia coli Synechoccus elongatus Escherichia coli Rhodopseudomonas acidophila Escherichia coli Escherichia coli Rhodospirrilum molischianum Mycobacterium tubercolosis Escherichia coli Bos taurus Potassium channel Photosyntetic reaction center Lactose permease G3P transporter Fumarate reductase Monotopic membrane protein Monoamine oxidase B Cyclooxygenase-2 Fatty acid amide hydrolase Squalene-hopene cyclase Coagulation factor V 1P7B 1PRC 1PV6 1PW4 1QLA Burkholderia pseudomallei Rhodopseudomonas viridis Escherichia coli Escherichia coli Wolinella succinogenes 1GOS 1CX2 1MT5 3SQC 1CZS Homo sapiens Mus musculus Rattus norvegicus Alicyclobacillus acidocaldarius Homo sapiens B C,D,E,G,J,K A A A A,B,C A A A,B,C A A A A B,F,I,J,L,M B,C A,B C,D A A,B A A A,B,C,D,G,I, J,K,L,M A H,L,M A A C Number of membrane helices 2 13 2 7 7 15 10 7 14 6 6 7 12 18 5 2 6 10 2 2 3 23 2 11 12 12 5 Number of interface helices 8 1 3 2 2 2 4 21 4 2 2 2 1 3 1 12 2 2 4 A A A A A The chain abbreviation is from the corresponding PDB structure. Tyr2 residues at G15 Å. The “positive inside” rule7 is also apparent, in that there is a clear increase in the frequency of Lys and Arg residues on the cytoplasmic side of the membrane. Asp and Glu, in contrast, seem to have no clear preference for any particular side of the membrane. The membrane–water interface region As indicated above, the parts of the proteins located in the membrane–water interface region G15–25 Å from the center of the membrane have distinct structural properties compared both to the parts located in the central region and those totally exposed to the aqueous phase. Lack of highresolution structural data has so far made it difficult to study this region, but the database of known structures is now big enough to make a first characterization possible. As shown in Figure 2(a), even when all transmembrane helices are removed there are still many hundreds of residues in the interface region. It is interesting to note that the total number of residues in successive 1 Å thick slabs is lower in the middle of the membrane than in the interface region (Figure 2(a), thick lines), indicating that helix bundle membrane proteins tend to have a “waist” near the center of the membrane. This waist is still seen even if cofactor-containing proteins are removed (data not shown). Figure 2(b) shows the distribution of secondary structure when the transmembrane helices have been removed. It can be seen that the interface region consists mainly of irregular structure (w70%) and interfacial a-helices (w30%). The helical content drops from G15 Å to G25 Å, while the fraction of irregular structure is maximal at G25 Å. There is a conspicuous lack of b-strand residues in the interface region, while further away from the membrane the b-strand content is similar to that of globular proteins.8 An example of a protein that contains many interfacial helices is the photosynthetic reaction center shown in Figure 3. The average length of interfacial helices resembles the average length of helices in globular proteins,9 while transmembrane helices are much longer, Table 3. The functional roles of interface helices are not well understood, and probably differ from protein to protein. The helices may be needed for structural reasons, such as to help positioning the DTD 5 ARTICLE IN PRESS Membrane–Water Interface of Membrane Proteins 3 Figure 1. (a) Distribution of secondary structure in the K50 to 50 Å region. (b) Distribution of amino acid residues. Hydrophobic residues are V, A, F, I, L, M, polar C, G, P, H, N, Q, S, T, aromatic W, Y, basic R, K and acidic D and E. The positive Z-coordinate is towards the periplasm/intermembrane side. Figure 2. (a) Amount of residues in 1 Å slabs before and after removal of transmembrane helices. (b) Distribution of secondary structure with transmembrane helices removed. The G12 Å region is removed due to small amounts of residues. ARTICLE IN PRESS DTD 5 4 Membrane–Water Interface of Membrane Proteins Figure 3. The photosynthetic reaction center (1PRC.pdb) chain M containing five TM helices (gray) and five interface helices (dark gray). The top of the picture is inside the cytoplasm and the C-terminal loop is not shown. The picture was made using PyMOL.29 transmembrane helices. In photosystem I, interface helices are thought to shield cofactors from the aqueous phase.10 Another functional role can be found in the MscS mechanosensitive channel, where an interfacial helix is thought to be involved in channel gating by transferring mechanical force from a “sensor” domain to a transmembrane helix in the ion channel.11 The KirBac 1.1 inwardly rectifying potassium channel also has an interface helix which is believed to slide parallel with the membrane in order to regulate the gating of the channel.12 Three main types of secondary structures Table 2. Distribution of amino acid residues in the G15–25 Å region Amino acid W Y C G P H N Q S T V A F I L M D E R K WCY CCGCPCHCNCQCSCT VCACFCICLCM DCE RCK Irregular structure (%) Interface helix (%) TM helix (%) 1.9 3.1 0.7 13.5 8.1 3.1 6.0 3.0 7.3 6.2 3.9 6.9 5.8 4.0 6.7 2.3 5.6 3.4 4.5 3.9 5.0 47.9 29.6 9.0 8.4 7.3 6.2 1.1 3.9 2.7 1.6 3.6 3.9 5.2 5.0 7.3 8.3 6.0 6.0 9.3 3.1 3.7 5.0 5.5 5.0 13.5 27.0 40.0 8.7 10.5 2.9 4.7 0.2 4.5 4.9 3.8 2.9 3.5 5.1 5.4 6.6 9.6 5.7 6.0 12.1 2.6 3.5 4.7 6.5 4.8 7.6 30.3 42.6 8.2 11.3 characterize the interface region: protruding ends of transmembrane helices, interface helices, and irregular structure. Table 2 shows that the three different types of interface structures differ in amino acid composition. The interface and transmembrane helices consist mainly of hydrophobic and polar aromatic (Trp, Tyr) residues; the latter are particularly abundant in the interface helices. Gly and Pro are enriched in the irregular structure, together with other polar residues such as Asn and Ser. These residues have been shown to be efficient turn-promoters when placed in the middle of long transmembrane segments,13 and also serve as helixbreaking or helix-capping residues in globular proteins.14 It has been reported that polar residues near the ends of transmembrane helices tend to point away from the center of the membrane, while apolar residues tend to point towards the center.15 We have carried out a similar analysis for the interfacial secondary structure elements by calculating the angle f of the Ca–Cb bond relative to the membrane normal for each residue in the interface region. Residues were classified into three categories: those pointing towards the membrane (fR1208), those pointing away from the membrane (f!608), and those parallel with the membrane (608%f!1208). The results are shown in Figure 4. Hydrophobic and aromatic residues tend to point towards the membrane, while polar and charged residues tend to point away from the membrane in all three classes of interfacial secondary structure. This tendency is strongest for the irregular structures, where 46% of the hydrophobic and 41% of TrpCTyr point towards the membrane. Two additional features are worth noting. First, the fraction of residues that are parallel with the membrane is highest for the TM helices (59%). Intuitively, sidechains of transmembrane helices should tend to be parallel with the membrane, since the helices are approximately perpendicular to the membrane plane. Second, the charged and polar amino acid residues in the interface helices are more frequently pointing away from the membrane (41–45%) than in the other secondary structure types (25–36%). The direction of the Ca–Cb bond is only a rough guide to the direction of long Tyr and Trp sidechains relative to the membrane plane, therefore we studied this in more detail using the angle between the Ca atom and the last polar atom on the sidechain. The Tyr and Trp side-chains tend to be directed towards the outside of the membrane when they are located below the membrane–water interface region around 15 Å; when located further out, they more frequently have their side-chains directed towards the membrane, see Figure 5(a). Interestingly, for Trp, this effect comes mainly from those residing in interface helices, 53% of the tryptophan residues that have their polar atom more than 1 Å closer to the lipid bilayer than their Ca atom are from interface helices. The turning of the polar group of Tyr in the 15–20 Å region, on the other hand, does not come from tyrosine residues in DTD 5 ARTICLE IN PRESS Membrane–Water Interface of Membrane Proteins 5 Figure 4. Direction of Ca–Cb for residues in interface helices, transmembrane helices and in irregular structures in the 15–25 Å region. interface helices, but from those in irregular structures which makes up 44% of those that are directed towards the middle of the membrane. Lys and Arg have long aliphatic side-chains with a positively charged amine or guanidinium group at the end. The aliphatic hydrocarbon part prefers to be localized in the hydrophobic region of the bilayer while the positively charged group prefers the more polar interface region, something described as snorkeling.16,17 The snorkeling effect of Lys and Arg can be observed, since the charged moiety of the side-chain in the 10–15 Å region is significantly higher than further away from the membrane region, see Figure 5(b). This effect is most prominent for Lys, with an average snorkeling of the polar atom of 2.5 Å, compared with 1.6 Å for Arg. The acidic Asp and Glu showed no statistically significant snorkeling, but were most often directed away from the membrane. Interface helices have a lot in common with amphipathic peptides residing in the membrane– water interface region. Snorkeling of Arg and Lys is a means of allowing the peptide to penetrate deeper inside the membrane and subsequently more strongly bound to it.18 Trp is capable of forming hydrogen bonds with its NH group, but it also has the largest non-polar surface of all amino acid residues. Tryptophan residues inside the membrane prefer to direct the polar NH group towards the aqueous region, while those in interface helices prefer to direct the NH group towards the lipid headgroup. This might lead to the possibility of burying the more hydrophobic six-membered ring inside the hydrophobic bilayer. Recently, Chamberlain et al.15 made a similar study of the snorkeling behavior of different amino acid residues in transmembrane helices in the 0–15 Å region. The above results are in agreement with their study, but also extend it to the 15– 25 Å region. The most striking result of the extension of the studied region is the change of direction of the polar atoms of Trp and Tyr when they enter the membrane–water region, and that the snorkeling of Lys and Arg occurs mainly in the 10–15 Å region. Loops in the interface region are responsible for connecting TM helices and interface helices. As seen in Table 3, the average lengths of loops between TM helices and interface helices are on average two residues shorter than those connecting two TM helices. It is rare (only seen 14 times, nine of them occurring in 1JB0) for an interface helix to be followed by another interface helix. Most interface helices thus connect two TM-helices. Longer loops connecting two transmembrane helices often contain interface helices, see Figure 6. The shortest loop containing an interface helix is eight residues, allowing for one turn of the helix and two short loops, but the majority are significantly longer, on average 27 residues, see Table 3. The longest loop without any interface helix is 31 residues, but most loops connecting two TM helices are short, on average nine residues and two-thirds are shorter than ten residues. In contrast, an DTD 5 ARTICLE IN PRESS 6 Membrane–Water Interface of Membrane Proteins Figure 5. Average distance between Ca atom and polar atom for different regions for (a) Trp and Tyr and for (b) Arg and Lys. A positive value means that the side-chain is directed away from ZZ0. The error bars are the standard error. Table 3. Average lengths (residues) for secondary structures close to the interface helix Secondary structure category Loops between helices TM helix–loop–interface helix TM helix-loop–TM helix Interface helix–loop–interface helix Helices Interface helix TM helix Length between TM helices Loop Interface helix Average length Min. length Max. length 7.0 9.0 8.6 1 1 1 35 31 19 8.9 26.0 4 15 19 43 9.0 27.2 1 8 31 69 interface helix between two TM helices separates the helices by more than 31 residues one-third of the time. As seen in Figure 6, long loops do not imply that the helices are spatially distant. In fact, for loops shorter than 20 residues, it is always loops lacking interface helices that separate the TM helices the most. For longer loops, the separation of TM helices varies significantly. Thus, interface helices might play a role as anchoring points for the positioning of the transmembrane helices. Monotopic membrane proteins Monotopic membrane proteins are soluble proteins or protein domains that bind to the surface of the membrane. As a comparison, we have analyzed four proteins listed as monotopic in the Membrane Protein Resource† plus the C2 domain of human coagulation factor V.19 The latter has a b-sandwich fold and contains b-strands and short connecting loops near its membrane-binding surface. The other monotopic proteins all have amphiphilic a-helices residing in the proposed membrane-binding interface.20–22 Fatty acid amide hydrolase and monoamine oxidase B both contain one predicted transmembrane helix but have their active sites and most of their mass in a globular, membraneinteracting domain (see the Membrane Protein Resource for further details). An examination of the amino acid distribution in the membrane-interacting regions (as defined in the original publications) reveals that the † http://blanco.biomol.uci.edu/MemPro_resources. html ARTICLE IN PRESS DTD 5 7 Membrane–Water Interface of Membrane Proteins Figure 6. Figure showing the distance (in Å) between the end/beginning of two TM helices against the number of residues connecting them. Filled squares are loops containing interface helix and small filled circles are loops without interface helices. membrane-binding parts of monotopic proteins by and large are similar to the interface structures discussed above. Some details are different, however. Acidic amino acid residues are half as common in the interface regions from the monotopic proteins (4% versus 9% in the transmembrane proteins) and basic residues are 50% more common compared to the interface structures in integral membrane proteins (15% versus 10% in the transmembrane proteins), strongly, suggesting that electrostatic interactions with negative charged lipid headgroups help stabilize membrane association.23 The most common amino acid in the interface regions from the monotopic proteins is Leu, which alone makes up 21% of the residues. The distribution of the Ca–Cb angles relative to the membrane normal show that most of the amino acid residues lie parallel with the membrane with the exception of Trp and Tyr (data not shown). In all, 64% of the Trp and Tyr residues are directed towards the membrane, which is a considerably higher fraction than for the interfacial structures in helix-bundle proteins, see Figure 4. This further emphasizes the importance of Leu, Trp and Tyr as membrane anchors, since monotopic proteins lack the rigid anchoring that transmembrane helices provide. interface region imposes clear structural constraints on integral membrane proteins. Of the residues in this region, w70% have irregular secondary structure, and the remaining 30% form interfacial a-helices oriented roughly parallel with the membrane plane. There is essentially no b-structure in the interface region. Irrespective of the type of secondary structure, aliphatic and aromatic residues tend to point towards the center of the membrane while polar and charged residues tend to point away from the center. The same general features are seen in the membrane-binding parts of monotopic membrane proteins, suggesting that common evolutionary forces shape all different kinds of protein surfaces exposed to the membrane– water interface. Materials and Methods Data set and analysis of known 3D structures PDB files for helix bundle membrane proteins of known structure were downloaded from the Membrane Protein Structure database†24 and directly from the Protein Data Bank.25 No chain with more than 30% sequence identity to another was included, resulting in a total of 56 nonhomologous chains from 27 PDB files, Table 1. To orient all membrane proteins, the first three and last Conclusions In summary, we find that the membrane–water † http://blanco.biomol.uci.edu/MemPro_resources. html ARTICLE IN PRESS DTD 5 8 Membrane–Water Interface of Membrane Proteins three Ca atoms from each TM helix in the biological unit defined a vector. These vectors were then used to calculate an average vector. The structure was finally rotated so that the Z-axis corresponds to this average vector and was therefore perpendicular to the plane of the membrane. The positive direction of the Z-axis was directed towards the periplasm/intermembrane side. The GES hydrophobicity scale26 was used to generate a hydrophobicity profile for each protein by first calculating the mean hydrophobicity for all residues with their Ca Z-coordinate located within the same 1 Å slice perpendicular to the membrane and then applying a 5 Å wide hat-shaped smoothing window. Thus, for a slab at Z-coordinate i, the smoothed average hydrophobicity profile was calculated as: hHðiÞi ¼ 1 ð3hðiÞ þ 2ðhði K 1Þ þ hði þ 1ÞÞ 9 2. 3. 4. 5. 6. þ hði K 2Þ þ hði þ 2ÞÞ The ZZ0 plane was defined as the minimum on the resulting hydrophobicity profile. The same kind of averaging was used for making Figures 1 and 2. Secondary structure classifications were made using STRIDE.27 Helices were re-classified as irregular structure if they were shorter than three residues and not flanking any other helix. One-residue b-bridges were also reclassified as irregular structure. V, A, F, I, L and M were classified as hydrophobic, C, G, P, H, N, Q, S and T as polar, W and Y as polar aromatic, R and K as basic, and D and E as acidic. Residues were classified into three categories: those pointing towards the membrane (fR1208), those pointing away from the membrane (f!608), and those parallel with the membrane (608%f!1208). The polar atom used for snorkeling analysis was Nz for Lys, N3 for Trp, Oh for Tyr and the mean coordinate of N3, Nh1 and Nh2 for Arg. The structures used for the analysis of the monotopic membrane proteins are listed in Table 1. Each structure was rotated using the Chimera visualization tool28 to have its Z-axis oriented perpendicular to the suggested membrane surface given in the corresponding articles. A hydrophobicity profile was then calculated for each protein in the same way as described above for the integral membrane proteins, and the protein was translated along the Z-coordinate until its hydrophobicity profile matched the averaged profile for the integral proteins as well as possible. Only one subunit of each of the structures was used for analysis and each chain had less than 30% sequence identity to each other. Amino acid distributions and side-chain directions were calculated as described above. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Acknowledgements 18. This work was supported by grants from the Swedish Natural Sciences Research Council and the foundation for Strategic Research to A.E. and G.vH. 19. References 20. 1. Wallin, E. & von Heijne, G. (1998). Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7, 1029–1038. Wallin, E., Tsukihara, T., Yoshikawa, S., von Heijne, G. & Elofsson, A. (1997). 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