10658
J. Phys. Chem. 1995,99, 10658-10666
Hydrophobic Nature of Membrane-Spanning a-Helical Peptides As Revealed by Monte Carlo Simulations and Molecular Hydrophobicity Potential Analysis Roman G. Efremov*V+** and Gerard Vergoten' Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, U1. Miklukho-Maklaya, 16/10, Moscow V-437, I1 7871 GSP, Russia, and Universiti des Sciences et Technologies de Lille, Centre de Recherches et d'Etudes en Simulations et Modilisation Moliculaires, UFR de Chimie, Bbtiment C8, 59655 Villeneuve d'Ascq, Cedex, France Received: October 13, 1994; In Final Form: February 23, 1995@
We propose an approach to explore the spatial hydrophobic and hydrophilic properties of transmembrane a-helical peptides. The computational procedure employs two independent techniques-statistical mechanics Monte Carlo (MC) simulations of nonpolar (propane) and polar (water) solvents around the peptides and three-dimensional molecular hydrophobicity potential (3D MHP) calculations. In the first approach, the polarity of a helix exposure was analyzed in terms of the average peptide-solvent interaction energies, whereas, in the second one, it was assessed using the 3D MHP distribution on the helix surface. The results obtained in the frameworks of both formalisms are in reasonable agreement, compliment each other, and provided a detailed presentation of the spatial hydrophobic nature of the peptides. Particular emphasis was put on testing the validity of simulations, examination of the convergence of energies in the MC runs, and comparison of the hydrophobicity measures obtained using different techniques. The method was applied to several transmembrane a-helical peptides from proteins with known structure. Resulting hydrophobic characteristics were compared with experimentally observed lipid- and protein-exposure of these segments in 3D structures of the membrane bundles. The approach was also employed in the hydrophobic mapping of putative channelforming a-helical peptide in epithelial amiloride-sensitive Na+ channel, and the results obtained were used to predict the residues lining the pore as well as exposed to a nonpolar environment. Future applications of the method to spatial arrangement of a-helices in membrane protein domains are discussed.
Introduction The membrane-embedded a-helical bundles have been identifiid in many proteins. The spatial arrangement of the helical segments inside a lipid bilayer may play an important structural and functional role. Such assemblies are stabilized by peptidepeptide, peptide-lipid, and, in a case of peptides forming ion channels, peptide-water interactions. Despite considerable efforts, three-dimensional (3D) structures of only a few membrane-spanning a-helical bundles in integral membrane proteins are known today.'J This lack of data is due to enormous difficulties in preparation of samples suitable for subsequent X-ray or electron microscopic analysis. A complementary approach lies in prediction of the spatial structure of intramembrane moieties using computer-aided molecular modeling. But even in the simplest case, if we will assume that the membrane-bound part is organized in the form of parallel or antiparallel a-helices, their mutual arrangement is still far from resolved. The main difficulty is that the forces driving self-association of peptides in the strongly nonpolar lipid environment are still not clearly u n d e r s t o ~ d . ~Most - ~ probably, helix-helix association is determined by both nonspecific protein-protein and protein-lipid binding as well as highly specific interactions between transmembrane (TM) peptides6
* Author to whom correspondence should be addressed. Present address: UniversitC des Sciences et Technologies de Lille, Centre de Recherches et d'Etudes en Simulations et ModClisation MolCculaires (CRESIMM), UFR de Chimie, Bitiment C8, 59655 Villeneuve d'Ascq, Cedex, France. ' Russian Academy of Sciences. Centre de Recherches et d'Etudes en Simulations et ModClisation MolCculaires. @Abstractpublished in Advance ACS Absrructs, June 1, 1995.
Analysis of well-resolved structures of membrane domains indicates that hydrophobic interactions are of prime importance for their structural organization. Thus, the residues exposed at the lipid face of an a-helical bundle are significantly more hydrophobic than those buried inside the protein, whilst the hydrophilic side chains of the helices line the interior of the bundle and helix-helix interfaces. Therefore, in order to develop molecular models of membrane-spanning domains, it is essential that the hydrophobic nature of individual TM a-helices is examined in some detail. Previous attempts to explore the hydrophobic character of TM helices employed visualization of helices as wheel plots,s calculations of hydrophobic moment^,^,'^ or Fourier-transform amphipathic analysis." Whilst such methods are able to delineate periodicity in the polarlnonpolar residue distribution along the sequence, more detailed information is needed to analyze the hydrophobic nature of TM segments as well as to orient them properly in the helix bundle^.'^^'^ A number of techniques have been employed to overcome this difficulty and investigate the spatial hydrophobic/amphipathic properties of TM a-helical peptides. Thus, Brasseur14 applied the 3D molecular hydrophobicity potentialIs (3D MHP) approach to differentiation between various types of lipidassociating helices. The formalism of 3D MHP utilizes a set of atomic physicochemical parameters evaluated from octanolwater partition coefficients (log P ) of numerous chemical compounds.Is It permits detailed assessment of the hydrophobic and/or hydrophilic properties of various parts of the molecules. Previously, we have developed a 3D MHP-based molecular modeling approach to analyze the spatial hydrophobic properties of membrane-embedded peptides and to calculate the interlocation of membrane-transversing helical hairpins.I6-l8 This
0022-365419512099-10658$09.00/0 0 1995 American Chemical Society
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Nature of Membrane-Spanning a-Helical Peptides
TABLE 1: Transmembrane a-Helical Peptides Used for Monte Carlo Simulations in Propane no. of propane a-helical no. of molecules dimensions of the box (A3) peptide residues residues" in the box A-BRhb 10-29 20 895 43.0 x 51.1 x 66.8 M3-PRC' 23 891 46.9 x 44.2 x 72.1 144-166 H1-GpAd 13-95 23 1057 48.2 x 48.1 x 14.2 M2-arENaCe 590-612 23 741 45.9 x 39.6 x 69.6 Without terminal acetyl and N-methyl groups. BRh = bacteriorhodopsin. PRC = photoreaction center of R. viridis.d GpA = glycophorin A. e Putative a-helical segment M2 in the a-subunit of the epithelial amiloride-sensitive Na+ channel. procedure provided a pictorial two-dimensional (2D) representation of nonpolar and polar patterns on the helix surfaces. In order to determine the polar face of TM channel-forming a-helical peptides and construct a number of initial channel models which can be used in subsequent simulations of the bundles, Sansom et used hydrophilic surface maps of the helical segments. The procedure employed 3D MHP analysis and empirical energy calculations of interactions of a peptide with a water molecule placed on a cylindrical polar grid around the peptide. Precise Monte Carlo (MC) simulations of water around a model polyalanine a-helix were performed in refs 20-23. In addition, such calculations were also done for uncharged solute and/or solvent molecules.22 A large number of the water shell configurations was analyzed to assess the relative contributions of hydrophobicity, packing, and proteinwater hydrogen bonding in structuring the solvent in the vicinity of the helix. Resulting radial distribution functions for solvent interactions and energetics of protein-water and water-water interactions revealed marked differences in hydration of polar (carbonyl oxygen) and nonpolar (Ca, Cp atoms) sites of the helix. Moreover, as it was shown for capped undecaalanine peptideFO the a-helix is more stable in water than the 310 conformation. Therefore, although the microscopic nature of the hydrophobic effects is still not completely understood, a number of conceptually different approaches have been employed to assess polarhonpolar properties of TM peptides. At the same time, no attempts were made to compare the results obtained in these techniques or to delineate their compatibility as well as possible discrepancies. Such analysis is believed to be useful in molecular modeling studies of protein folding (including association of Th4 peptides in the membrane domains) and stability. With this aim in view, here an attempt is made to apply two different formalisms-statistical mechanics MC simulations of nonpolar and polar solvents around a helical peptide as well as a 3D MHP calculation in order to characterize the spatial hydrophobic properties of TM a-helices. Assessment of hydrophobicity maps of individual peptides with their spatial arrangement in real 3D structures of membrane proteins was done to explore the nature of the hydrophobic organization of intramembrane a-helix bundles. al.'39'9
Method of Calculation As summarized in Tables 1 and 2, the following transmembrane a-helical segments were used in this study: helix A in bacteriorhodopsin (BRh) (entry lBRD in the Brookhaven Protein Data Bank, PDB24);segment M3 in the photoreaction center (PRC) of R. viridis (entry lPRC in PDB); the TM segment in human glycophorin A ( G P A ) ; and ~ ~ the putative TM segment M2 in the a-subunit of the rat epithelial amiloridesensitive Na+ channel (rENaC).26 For the membrane-spanning
TABLE 2: Transmembrane a-Helical Peptides used for Monte Carlo Simulations in HzO no. of H20 a-helical no. of molecules dimensions of the box (A3) peptide residues residues" in the box A-BRhb 10-29 20 2071 31.6 x 39.4 x 54.5 M2-arENaC' 590-612 23 2996 42.3 x 35.0 x 66.2 H1-GpAd 13-95 23 2986 38.7 x 39.2 x 63.8 Without terminal acetyl and N-methyl groups. BRh = bacteriorhodopsin. Putative a-helical segment M2 in the a-subunit of the epithelial amiloride-sensitive Na+ channel. GpA = glycophorin A. (I
peptides with unknown 3D structure, initial helical geometries were generated using standard &J/?/Jangles and side chain conformations that occur most frequently in a-helices of known protein structures. In all peptides, acetyl (Ace) and N-methyl (N-Met) groups were attached to N- and C-termini, respectively. Before the solvent simulations, TM helices with added hydrogens were subjected to 100 cycles of steepest descents minimization (with fixed heavy atoms (except Ace and N-Met) in the segments from BRh and PRC). Then, the peptides were minimized without constraints, using 3000 conjugate gradient steps. This was done with the Discover program and CVFF force field.27 MC simulations of a-helical peptides placed in the rectangular boxes of nonpolar (propane) and polar (TIP3P water) solvents were implemented using the BOSS program?8 The united-atom OPLS force field parametersz9 were used to represent intermolecular interaction energies by Coulombic and Lennard-Jones terms. The dimensions of the solvent boxes (Tables 1 and 2) were chosen to provide a layer of solvent around each peptide atom at least 11-12 8, thick. The nonbond interactions were truncated at 10 8,. All calculations were performed for systems with periodic boundary conditions in the isothermal-isobaric (NPT) ensemble at 25 "C and 1 atm. In some cases, in the beginning of equilibration, the NVT ensemble was also used. The systems were equilibrated for (1 -0- 1.5) x lo6 configurations until the convergence of the total energy and the energy of the solute-solvent interaction was achieved. Other details of MC simulations can be found in ref 28. To analyze the results, we used a set of programs specially written for this. In the first stage, the energies of interaction between each protein atom and surrounding solvent molecules were calculated by averaging over a set (usually 30-50) of configurations extracted with intervals of 1 x lo5 MC steps. For such an analysis performed in propane, we accounted for only the protein atoms in the central part (30 8, thick) of the helical segment which corresponds to the pure hydrocarbon core of the lipid bilayer.30 For simulations in water, the edges of the segments were also taken into consideration. For the most pictorial presentation of nonpolar and polar properties of a-helical segments, we calculated the angular distribution of these atomic energies. Initially, the coordinates of the protein atoms were transformed into a coordinate system (Figure 1) with axis Z corresponding to the helix axis (calculated using least-squares fit to backbone atoms) pointed from the C- to N-terminus and axis X passing through the C, atom of the N-terminal residue (except Ace). The polar angle a was counted in an anticlockwise direction from axis X when looking downward along the Z axis. In the next step, we sum the atomic solute-solvent interaction energies inside a sector with angular size 90" slicing with angular increment 5" or 10". For each sector, the total solutesolvent interaction energy was attributed to the central angle of the arc. The resulting dependence of the intermolecular interaction energy on the rotation angle was used to detect the
10660 J. Phys. Chem., Vol. 99, No. 26, 1995
A Z J
Figure 1. The scheme illustrating a coordinate system ( a , 2)used for presentation of the spatial hydrophobic properties of TM a-helices. Axis 2 is coincident with the helix axis, and a is the rotation angle subtended in the plane perpendicular to axis 2. Axis X passes through the C, atom of the N-terminal residue.
nonpolar and polar sides of the TM a-helical segments. The same approach was also applied to calculate the angular dependence of 3D MHP on the peptide surface. The 3D MHP created by protein atoms (including hydrogens) in the surface points of helical segments was calculated and visualized by means of 2D plots in coordinates (a, z), where a is the rotation angle (see above) and z coincides with the helix 2 axis. This was done as described earlier.16 Moreover, for segment A in BRh, we calculated the 3D MHP created by surrounding propane molecules on the peptide surface. In order to do this, we added hydrogens to the system “helix i-propane” obtained as the result of MC simulations and subjected it to 100 cycles of steepest descent minimization with fixed heavy atoms. This was done using the Discover program and CVFF force field.27 The resulting configuration of the system was used to calculate the 3D MHP on the peptide surface. Atomic hydrophobicity constants for peptide and propane molecules were taken from ref 31.
Results and Discussion 1. Monte Carlo Simulation Of Nonpolar Solvent and Water around a-Helical TM Peptides. A number of approximations were used in order to perform the calculations and simplify the analysis. (i) Computer-built conformations of
Efremov and Vergoten a-helical peptides for proteins with unknown spatial structure can be somewhat different from their native ones. At the same time, the main idea of this work is to investigate preferences of lipid- and interior-exposure of membrane-spanning peptides. Therefore, slight differences in the helix conformation are believed not to change significantly the results. Thus, as we discussed earlier,l6 the spatial hydrophobic properties of several experimental and computer-generated TM helices in the PRC of R. viridis were found to be in good agreement with each other. (ii) The internal coordinates of the helices were kept rigid during Monte Carlo runs and only the solvent molecules were allowed to move. Such an assumption was investigated in detail in ref 21 by comparing the results of MC simulations of a rigid helix and those obtained by molecular dynamics of a partially mobile helix in a water box. No significant differences in the distribution and orientation of water molecules were seen, indicating that neither the box side, united atom representation, nor rigidity of the helix affected the results meaningfully. This should be particularly true for the systems considered here, a-helices in propane, due to the absence of peptide-solvent hydrogen bonding. To investigate in more detail the dependence of surface hydrophobicity maps on the side chain flexibility, we have carried out molecular dynamics simulations of TM helices and analyzed 2D MHP maps for several families of lowest-energy conformers (Efremov & Vergoten, in preparation). The main conclusion is that the spatial hydrophobic characteristics of such peptides are well preserved (especially in their central parts) during dynamics runs. (iii) Simulations of nonpolar solvent (propane) were performed for full TM helices although their terminal parts in the real membranes can strongly interact with polar head groups of lipids and water molecules on the water-bilayer interface. To overcome this limitation, in the analysis of angular distributions of solute-solvent interaction energies, we accounted for only residues in the central part of TM segments (30 A thick) which to the hydrocarbon core Of the 1.1. Convergence of the Total Energy and the Energy of Peptide-Solvent Interaction during MC Runs. MC simulations of peptides 20-25 residues long placed in a box of solvent are time-consuming and should include long-length equilibration of the system. Therefore, a choice of the reasonable and short enough length of equilibration is useful to reduce a CPU time. With this aim in view, we analyzed the convergence of different parameters of the system (total energy, solute-solvent interaction energy, volume, heat capacity, etc.) with a number of MC iterations. Typical dependences of the total energy as well as the energy of solute-solvent interaction on the number of steps are shown in Figure 2A and B for a-helical segment A of BRh in propane and water, respectively. It is seen that the peptidepropane interaction energy converges already after (4-6) x lo5 configurations. Calculations of even shorter lengths were shown to provide convergence of the total energy for this system (Figure 2A, inset). As seen in Figure 2B, MC simulations of a-helices in water are considerably more expensive than those in propane. Approximate convergence of energetic parameters was achieved only after 1 x lo6 steps of equilibration. This is mainly due to strong water hydrogen bonding to the peptide, which is accompanied by significant fluctuations of the total energy and the energy of solute-solvent interaction. It should be noted that thermodynamic quantities of the second order, like heat capacity, compressibility, etc., converged significantly more slowly. For the essentially qualitative calculations performed here, it is felt this computational protocol is reasonable. The main goal we pursued in this study is not to
Nature of Membrane-Spanning a-Helical Peptides
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Figure 3. Angular distributions of interaction energies (ESS) between helix A in bacteriorhodopsin and propane for seven consequent Monte Carlo configurations. N = 0 corresponds to the last configuration obtained during equilibration of the system. Spacing between configurations: 5 x lo5 steps. Atomic solute-solvent interaction energies are summarized inside the sector 90O-width. Rotation angle is defined as in Figure 1.
-300.0 1
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Figure 2. Monte Carlo simulation of propane (A) and water (B) around a-helix A in bacteriorhodopsin. Dependence of peptide-solvent interaction energy (ESS) on the number of Monte Carlo steps (N). Insets: total energy of the system vs the number of Monte Carlo steps. Values along axis X are given on the logarithmic scale.
obtain exact thermodynamic properties of such systems but to explore the general tendencies in polar and nonpolar exposure of membrane peptides. Considering this as well as taking into account the approximations inherent to the method, we have found that reliable results of MC simulations in C3Hg can be achieved after about 4 x lo5 steps of equilibration followed by 3 x lo6 steps of data averaging. 1.2. Angular Dependence of Peptide-Solvent Interaction Energies. Because of the symmetry of a helical peptide, useful results on its polarhonpolar exposure can be obtained from the plots presenting dependence of hydrophobicity parameters on the rotation angle around the helix axis (a). Below, for such a kind of presentation we will use the term "angular distribution plot". In the present study, the analysis of solvent molecule distributions obtained as the result of MC simulations was focused on the angular dependence of atomic peptide-solvent interaction energies averaged over a large enough set of configurations. In order to test how the results depend on the number of MC steps, we analyzed the angular distribution plots obtained for simulations of helix A of BRh in propane (Figure3). The plots were calculated for several molecular configurations extracted with intervals of 1 x lo5 MC steps after equilibration of the system. As seen in Figure 3, all the curves are quite similar even for configurations separated by two million iterations. The important conclusion was made that the pattems of atoms and residues revealing the strongest interactions with nonpolar solvent can be established as a result of relatively short MC runs (about 1 x lo6 steps). Further increasing of the number of steps does not lead to significant changes in the distribution
of solvent molecules around the peptide and the energetics of the peptide-solvent interaction. MC simulations in water included at least (1.5-2.0) x lo6 steps of equilibration and (12) x IO6 steps of data averaging. As we will demonstrate below, almost all hydrophobic TM helices studied in this work reveal prominent pattems of strong and weak interaction with nonpolar solvent. On the contrary, angular distribution plots obtained as a result of water simulations are noisy due to the presence of protein atoms involved in hydrogen bonding with water. Qualitatively the same effect also was observed for polyalanine helix simulations in "normal" and uncharged water.** As follows from the definition of united-atom OPLS parameters for amino acid residues and propane,29peptide-propane interactions are described only by means of the Lennard-Jones term. This leads to tight packing of propane molecules around the peptide. On the other hand, the TIP3P model of water also accounts for electrostatic interactions responsible for hydrogen bonding. According to the results obtained in ref 22, hydrogen bonding plays a dominant role in helix-water interactions. Due to the highly directional character of hydrogen bonds, small variations in the orientation of waters with respect to protein donor or acceptor groups induce significant changes in peptide-water interaction energies. That is why the variation in protein-solvent interaction energies is much less in MC simulations in propane than in water. More extended discussion about this can be found in ref 22. In this study, the angular preference in peptide exposure to water was observed only for strongly amphiphilic peptides like the putative pore-forming a-helix M2 in the a-subunit of rENaC (Figure 4A, solid line). This peptide contains three negativelycharged residues (Glu595, 598, and Asp602) lying on the same helix side, with 190" < a < 300". The energy minimum is clearly visible, corresponding to favorable interactions of these residues with water. At the same time, nonamphiphilic, sufficiently hydrophobic TM peptides (like helix A in BRh, Figure 4B, solid line) do not demonstrate prominent maxima and minima in the angular distribution of protein-water interaction energies. I .3. Correlation between peptide-solvent interaction energies and 3 0 MHP. The angular distribution of atomic energies of
10662 J. Phys. Chem., Vol. 99, No. 26, 1995
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t
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Figure 4. Monte Carlo simulation of water around a-helical peptides. (A) Putative TM a-helical segment M2 in amiloride-sensitive Na+ channel. (B) TM a-helical segment A in bacteriorhodopsin: (solid line) angular distribution of peptide-water interaction energies (ESS); (dotted line) angular distribution of 3D MHP on the peptide surface.
interaction between the a-helical segment and surrounding nonpolar and polar solvents, provides a pictorial representation of spatial hydrophobic and hydrophilic properties of the peptides. It is interesting to compare such characteristics with those obtained in complementary approaches. Previously, we have found that the spatial polarity of membrane-spanning helices can be expressed in terms of 3D MHP calculated at the surface points of these peptides.I6 Figure 5 shows a 2D contour map of MHF’ on the surface of helix A in BRh. Only the hydrophobic areas with high values of MHP are indicated. The angular distributionsof surface 3D MHP and energy of peptidepropane interactions are depicted on the bottom of Figure 5 with dotted and solid lines, respectively. These plots demonstrate strong anticorrelation (correlation coefficient, r = -0.88), c o n f i i n g that the most prominent hydrophobic surface regions (high values of MHP) strongly interact with nonpolar solvent revealing the lowest energies of peptide-propane interaction. Obviously, angular distribution plots give a picture of polar and nonpolar sides averaged over the helix length. In order to compare in more detail these measures of hydrophobicity, we superimposed the peptide atoms with the lowest energies of interaction with propane and with water (averaged in a set of MC configurations) upon the 2D MHP map (Figure 5, top). In the following, we will use the term “2D ESS map” for such a type of atomic projections. It is seen that almost all the atoms strongly interacting with nonpolar solvent fall into the areas with large positive values of MHP or are located close to their boundaries. On the contrary, the atoms revealing low energies of interaction with water are mainly distributed outside these areas. The exceptions are provided by the tryptophan residues 10 and 12 and methionine 20: some of their atoms with low energies of peptide-propane interaction lay outside the areas of high MHP. The comparison of 2D MHP maps and 2D projection maps of the atoms most favorably interacting with propane and water was also done for the other TM segments under study (see
I
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0
,
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,
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I
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Y
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,
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Figure 5. Spatial hydrophobic properties of the membrane-spanning a-helical segment A in bacteriorhodopsin. (top) Two-dimensional isopotential map of the molecular hydrophobicity potential (MHP) on
the peptide surface. The value on the X axis correspondsto the rotation angle about the helix axis; the parameter on the Y axis is the distance along the helix axis. Only the areas with MHP > 0.1 are shown. Contour intervals are 0.02. The positions of residues are indicated by letters. The symbols * and A represent projections of peptide atoms revealing the lowest energies of peptide-propane (ESS < -0.9 kcaymol) and peptide-water (ESS -30 kcaymol) interactions, respectively. The most variable side of the segment calculated by the method’ is labeled with an arrow. Filled bars and dashed lines represent lipid exposure of the segment in the experimental structure.33(Bottom)Solid line, angular distribution of peptide-propane interaction energy (ESS); dotted line, angular distribution of MHP on the peptide surface. ESS and MHP are summarized inside the sectors 90O-width. Figures 6-8, top). Their hydrophobic properties will be considered in more detail in the subsequent discussion. Here we would like to point out the main inferences made from such analysis. All the helices reveal a good correlation between 2D distributions of their nonpolar/polar characteristics. This conf m that the energies of peptide-solvent interaction and surface 3D MHP provide a qualitatively similar description of spatial hydrophobic properties of helical segments. At the same time, exceptions also occur. Thus, some atoms in residues N147 and H162 in TM segment M3 from PRC (Figure 6, top), G94 in GpA (Figure 7, top), and M610 and L612 in helix M2 from a-rENaC (Figure 8, top) demonstrate low values of ESS but do not create strong positive MHP in the neighboring surface points. At the same time, a number of atoms in residues 1177, I191 in GpA and F601, V605 (helix M2, a-rENaC) exhibit low energies of peptide-water interaction but fall into the prominent hydrophobic areas on 2D MHP maps. Therefore, the results of MC simulations and MHP analysis often complement each other, providing an additional insight into the hydrophobic properties of TM peptides. In these cases both formalisms should be taken into account. It seems reasonable to assume that those several discrepancies found between MHP- and ESS-data can be corrected in future studies
Nature of Membrane-Spanning a-Helical Peptides
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Figure 8. Spatial hydrophobic properties of putative membranespanning a-helical segment M2 in the a-subunit of the epithelial amiloride-sensitiveNa+ channel. All the details are the same as in the
legend to Figure 5. by fitting of corresponding atomic hydrophobicities andor OPLS force field parameters to provide a mutually consistent picture of the spatial hydrophobic properties of TM segments. Thus far we considered only MHP created in the surface points by the protein atoms (MHPpmtein).In addition, we also calculated 3D M H P induced on the same surface by surrounding solvent molecules (MHP,o~vent). If we propose that solvation of a peptide in a strongly nonpolar solvent is driven mainly by hydrophobic interactions, it is reasonable to expect that the hydrophobic patches on the protein surface created by protein atoms and those induced by neighboring solvent should be disposed against each other. This should also be true for hydrophilic patterns. Here we have employed calculations of MHPsolvent for TM segment A in BRh. Exact positions of propane molecules around the helix were taken from MC data. Resulting plots of surface MHPprotein and MHPsolvent YS angle a are given in Figure 9. Both curves reveal very similar features and correlate with coefficient r = 0.97. Therefore, hydrophobic patterns on the peptide surface tend to be in more close contact with nonpolar solvent molecules than the hydrophilic ones. Note that the MHPsOlvent plot is more significantly shifted to positive values of MHP than that for MHPprotein.Obviously, this shift depends on what surface was chosen. Thus, reducing the probe radius in calculation of the Connolly surface will result in closer agreement between the absolute values of both plots while their shapes will be conserved. Because the main idea of this example was to assess spatial hydrophobic characteristics
c3
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Figure 6. Spatial hydrophobic properties of membrane-spanning .~~ a-helical segment M3 in the photoreaction center of R. v i r i d i ~All the details are the same as in the legend to Figure 5. Only the projections of peptide atoms revealing the lowest energies of peptide-propane
interactions are shown. obtained in complementary approaches, particular importance was attached to the distributions of relative values of 3D MHP. We should point out that the two formalisms applied here for description of peptide hydrophobicity are independent and based on different principles. Thus, OPLS force field parameters used in MC calculations were derived as the result of computer simulations, by a comparison of the experimental and calculated physicochemical properties of pure liquids and peptides in the crystalline On the other hand, atomic hydrophobicity constants implemented in the 3D MHP approach were extracted from the analysis of experimentally observed partition coefficientsof different organic compounds in octanolwater mixture^.^' Therefore, a good accordance between these two formalisms gives a strong argument that both approaches are able to provide a realistic description of the hydrophobic nature of TM peptides.
2. Spatial Hydrophobic Properties of,Membrane-Spanning a-Helical Peptides. We applied the computational protocol presented above to assess the spatial hydrophobic properties of two TM helical peptides taken from integral membrane proteins with known structure (BRh and PRC) and the TM segment in GpA-the system which provides a body of experimental evidence about the structurd organization of its
10664 J. Phys. Chem., Vol. 99, No. 26, 1995
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t
230
ments in BRh-this will be done elsewhere (Efremov & Vergoten, in preparation). We restrict the discussion to the case of a-helix A. The 2D contour map shown on the top of Figure 5 indicates areas with large values of MHP on the surface of helix A. The most prominent stretch of hydrophobicity is observed in the range of angle a = 135-270" and extends through the whole peptide length. It is formed by residues 11, 15, 19,22,26, and 27. Another hydrophobic zone corresponds to residues 10, 14, 25, 29 (310' < a < 360"), and 28 (0' < a < 50"). The strongest polar areas are attributed to residues 16, 21, and 24. The histogram of surface MHP for segment A (not shown) is significantly shifted to the high values of MHP. This is typical for the membrane-spanning peptidesI6 and indicates the strong nonpolar character of its surface. The plot of atomic peptidepropane interaction energies summarized in the arc with angular size 90" vs rotation angle a of the helix is shown in the bottom part of Figure 5 with a solid line. The prominent minimum corresponds to the helical face with 160" < a < 260'. Low energies are also observed for the regions 0-20" and 300360". The curve characterizing the angular distribution of surface MHP of this segment is presented with a dotted line. As was discussed above, both plots as well as 2D MHP and 2D ESS projection maps provide a very similar picture of nonpolar and polar exposure of the helix. (The exceptions presented by residues W10, W12, and M20 were also mentioned.) Analysis of the experimentally determined structure of BRh33 reveals that the lipid-exposed face of the segment contains residues 10, 11, 14, 15, 18, 19,22,25,26, and 29. The interface with helices B and G is formed by residues 12, 13, 16, 20, 24, 27, and 28. The first region corresponds to the intervals of a 170-360" and 0-30', whereas the second one corresponds to a = 30-180". Therefore, that side of the helix which was predicted to be the most hydrophobic is exposed to the bilayer in the BFth bundle. On the contrary, the most polar side is in contact with the other protein segments. Note that the most variable side of this TM segment (it is marked with an arrow in Figure 5 , top) is assigned as the strongest hydrophobic one on the 2D MHP map and on the angular distribution plots. Although this is believed to be a common feature for the membrane a-helix bundles? the exceptions are not rare. We will demonstrate this in our future article. 2.2. Helix M3 in the Photoreaction Center. The photoreaction center from R. viridis is the first integral membrane protein whose crystal structure was solved with atomic resolut i ~ n Its . ~membrane ~ domain consists of eleven TM a-helices belonging to L-, M-, and H-subunits. Five helices each in L and in homologous M chains form a two-layer bundle which is approximatively perpendicular to the membrane plane. For subsequent analysis we choose TM segment M3 which lies in the central part of the bundle. The 2D MHP map for helix M3 is shown in the upper part of Figure 6. As in the case of helix A in BRh, there is a prominent hydrophobic stretch on the peptide surface which spans all the helix length. It is formed by residues 1144, F148,1152, F153, L156,1161, and L165 and is tilted about 30" away from the 2 axis. Pronounced nonpolar areas are also observed in the vicinity of residues W146, F154, and V155. The angular profiles of peptide-propane interaction energy and 3D MHP (Figure 6, bottom) reveal the most hydrophobic face of the helix at values of a = 140-230" and 140-300", respectively. Note that, like for segment A in BRh, these plots exhibit prominent anticorrelation. Moreover, as seen
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'
I
,
60
,
.
,'
t
I
e
120
180
240
300
z
'O
30
360
ROTATION ANGLE, dag.
Figure 7. Spatial hydrophobic properties of the membrane-spanning a-helical segment in glycophorin A. The residues critical for the formation of dimer25are boxed. Dashed lines represent the boundaries of the putative helix-helix interface in the dimer. All the details are the same as in the legend to Figure 5.
0
60
120
180
240
300
380
ROTATIONANGLE, deg.
Figure 9. 3D MHP on the surface of a-helix A in bacteriorhodopsin: - MHP created by protein atoms; (dashed line) (solid line) MHPproteln MHP,,~,,.t - MHP created by surrounding propane molecules. Positions of propane molecules are obtained from the results of Monte Carlo simulations.
membrane domain. The main task we pursued here was to demonstrate how the spatial polarity of helices correlates with their arrangement in the membrane bundles. Finally, the approach was employed to hydrophobic mapping of the putative channel-fonning a-helical peptide in rENaC, and the results obtained were used to predict the residues lining the pore as well as those exposed to the nonpolar environment. 2.I. TM Helix A in Bacteriorhodopsin. Bacteriorhodopsin (BRh) is one of the best-characterized membrane proteins whose model is available with atomic resolution and consists of seven TM a-helices arranged in a kidney-shaped manner.33 The limited size of this paper makes it difficult to discuss in detail the hydrophobic nature of all seven membrane-spanning seg-
Nature of Membrane-Spanning a-Helical Peptides in 2D MHP and ESS maps, almost all the atoms with low peptide-propane interaction energies fall inside the areas of high MHP. This confirms a general agreement between two formalisms used to quantify spatial hydrophobic properties of the peptide. At the same time, some interesting exceptions are observed for polar side chains of residues N147 and H162: as was reasonable to expect, they do not create high MHP in the neighboring surface points while revealing favorable interaction with the hydrophobic solvent. This results in a small discrepancy between 1D MHP and ESS plots in the region of a = 60-100" (Figure 6, bottom). Inspection of the crystal structure of PRC34justifies that the lipid-exposed face of this helix includes residues A145, W146, A149,1152, F153, L156,1161, T164, and L165 and corresponds to values of a in the interval 130-270'. Therefore, this segment is oriented in the membrane bundle in such a way that its most nonpolar side faces to the bilayer, whereas less hydrophobic pattems are in contact with neighboring protein segments or tumed inside the bundle. 2.3. TM Helix in Glycophorin A. Glycophorin A (GpA) from human erythrocytes contains a single membrane-spanning a-helical anchor that mediates the formation of stable dimersz5 Multiple amino acid substitutions in this peptide led to identification of residues critical for the helix association. Coincident with mutagenesis experiments, the molecular modeling study implemented simulated annealing search of the best energetically-favored conformations was undertaken in ref 35. The results showed a high propensity for a right-handed supercoil and were in good agreement with mutation data. The simplicity of the system (two identical membranespanning a-helices) and availability of experimental data on its structural arrangement make the TM domain of GpA a suitable model for testing the algorithms destined for prediction of a-helix packing in membrane domains of proteins. As seen on the top of Figure 7, the surface of the TM a-helix in GpA is sufficiently hydrophobic, except the region close to the glycines and T87. Hydrophilic patches in the vicinity of the glycines are due to the close proximity of polar backbone atoms, whereas that near threonine is induced by its polar side chain. In the coordinate system adopted here, such a hydrophilic pattem corresponds to values of a between 30" and 150". It is clearly seen in the angular ESS and surface MHP plots (Figure 7, bottom) and corresponds to maximal energies of peptidepropane interaction as well as to minimal values of MHP. It is likely that this region can be involved in binding with another TM peptide and, thus, tends to be shielded from the nonpolar lipid environment. The most hydrophobic pattem on the surface is formed by residues 173, L75, 177, F78, 185, 188, and L89 and has coordinates 180" < a 300" (Figure 7, up). As follows from the analysis of ESS and surface MHP plots, this side of the helix demonstrates the highest potency to interact with nonpolar solvent and, thus, most probably, is exposed to the lipid surrounding. The other region which strongly interacts with the hydrocarbon environment includes residues V80, 191, and I95 (Oo < a < 30"). As discussed above, the discrepancy between 2D MHP and 2D ESS maps is mainly caused by the residue G94 (hydrophilic character according to MHP data and hydrophobic character according to ESS data) and some atoms in residues I77 and I91 (low energies of peptide-water interaction but high MHP on the surface). A model for the dimerization of the TM a-helix in GpA derived from mutationalz5and c~mputational~~ studies reveals a sequence motif L75-176-xr-G79-V80-xr-G83-V84-xr-T87 that
J. Phys. Chem., Vol. 99, No. 26, 1995 10665 drives helix association. As follows from the 2D MHP map (Figure 7), these residues are distributed in the interval of a = 30-160" and, therefore, lie on the less hydrophobic face of the helix, Interestingly, the hydrophobic and hydrophilic stretches on the peptide surface are not exactly vertical. If one assumes that in the dimer the strong and weak hydrophobic areas are facing outside and inside the bundle, respectively, and the segments are tightly packed through their hydrophilic surfaces, then interlocation of the helices should deviate from the parallel orientation. This is consistent with the model of the coiledcoil conformation proposed in ref 35. 2.4. Putative Channel-Fonning TM Helix in the Epithelial Amiloride-Sensitive Nu+ Channel. TM bundles of parallel or antiparallel helices may form ion channels in a cell membrane.36 The challenge in modeling such bundles resides in construction of a number of initial conformations for further refinement and analysi~.'~ In these assemblies, the hydrophilic side chains of the helices line the central pore, whilst the hydrophobic residues interact favorably with the fatty acyl chains of lipids. Therefore, knowledge of spatial hydrophobichydrophilic properties of channel-forming peptides may provide important constraints on their mutual arrangement in the bundle. In the subsequent discussion, we have employed the computational protocol described above, to assess the hydrophobic nature of the putative pore-fonning a-helix in the epithelial amiloride-sensitive sodium channel (rENaC).26 Recent experimental data26 as well as computational processing of the amino acid sequence of rENaC (Efremov et al., manuscript in preparation) provide a body of evidence that the peptide M2 (590-612) forms the membranespanning a-helix and participates in ion transport. The peptide does not reveal pronounced hydrophobic pattems on the surface which span its whole length. Therefore, this hampers the use of 1D angular distribution plots. In such a case, assessment of the spatial polarity can be made only on the basis of 2D maps (Figure 8, top). The most nonpolar areas are observed in the ranges 10" < a < 90" and 315" a < 360" (residues V590, V593, A597, 1600, F601, L604, L608, L611, and L612). Therefore, these regions exhibit a tendency to be exposed to the bilayer. Hydrophilic patches on the surface are located around residues S592, E595, E598, D602, and T607. As was reasonable to expect, all charged side chains create negative M H P in the neighboring surface points. Comparison of angular distributions of surface MHP and energies of peptide-water interactions (Figure 4A) obtained for helix M2 in a-rENaC reveals their good correlation ( r = 0.78). As we already mentioned above, deep minima on both plots correspond to the helix side containing three negatively-charged residues. Moreover, 2D projection of the atoms most favorably interacting with propane and water (Figure 8, top) shows that most of them fall, respectively, inside and outside the areas of strong hydrophobicity. The exceptions are provided by residues S592, M610, and L612-some of their atoms with low values of ESS are disposed in the relatively polar areas on the 2D MHP map. In addition, some atoms with low values of peptidewater interaction energy (residues F601 and V605) are observed inside the nonpolar surface areas. Interestingly, in the central part of the segment, the polar surface regions are separated with a nonpolar stretch formed by residues V599, L603, 1606, and L609. One can propose that such a "belt of hydrophobicity" may be important for regulation of the ion transport through the channel. But in the absence of any additional data, this assumption remains quite speculative.
10666 J. Phys. Chem., Vol. 99,No. 26, 1995
Efremov and Vergoten
Conclusions
References and Notes
In the present study, we have proposed an empirical method for the assessment of the spatial hydrophobic and hydrophilic properties of TM a-helical peptides. The approach employs two different techniques, Monte Carlo simulations of nonpolar solvent and water around the peptide as well as analysis of 3D MHP on its surface. The results obtained allow the following conclusions: (i) The spatial hydrophobic characteristics of TM a-helical peptides assessed by the independent techniques are in reasonable agreement with each other. At the same time, several exceptions were found, thus confirming the complementarity of both formalisms in these cases. (ii) Resulting detailed mapping of peptide polarity and analysis of interactions between a-helices and surrounding solvents permit identification and pictorial visualization of the amphipathic nature of TM peptides. This can be done using both 1D-and 2D-representations in terms of angular distribution profiles as well as MHP andor solute-solvent interaction energy maps. (iii) Being applied to several TM a-helical peptides from integral membrane proteins with known structure, the method helps to explore the relationship between the hydrophobic properties of the helices and their spatial arrangement in the membrane-spanning bundles. (iv) Analysis of amphipathic nature of the putative TM a-helical segment in the epithelial amiloride-sensitive Na+ channel makes it possible to predict amino acid residues lining the pore and those exposed to the nonpolar surrounding. Current studies are centered about implication of the computational technique to assess the hydrophobic organization of a-helical domains in membrane proteins with known structure. Such an analysis is believed to gain a better insight to understanding the mechanism driving the formation of helix bundles in a lipid environment. Our future work is also being pursued to use this approach for modeling the ion channels (e.g. rENaC) which are built from highly amphipathic a-helical peptides and, therefore, impose strong restrictions on the disposition of their hydrophilic pattems. Finally, recent progress in site-directed mutagenesis of TM segments calls for estimation of the effects induced upon amino acid substitution on the structural and hydrophobic properties of the peptides. The technique proposed in this study may provide an essential help in such analysis.
(1) Cowan, S. W.; Rosenbusch, J. P. Science 1994, 264, 914. (2) von Heijne, G. Annu. Rev. Biophys. Biomol. Struct. 1994,23, 167. (3) Cramer, W. A.; Engelman, D. M.; von Heijne, G.: Rees, D. C. FASEB J. 1992, 6, 3397. (4) Lemmon, M. A.; Engelman, D. M. Curr. Opin. Struct. Biol. 1992, 2, 511. (5) Popot, J.-L. Curr. Opin. Struct. Biol. 1993, 3, 532. (6) Lemmon, M. A.; Engelman, D. M. FEBS Len. 1994, 346, 17. (7) Rees, D. C.; DeAntonio, L.; Eisenberg, D. Science 1989,245,510. (8) Schiffer, M.; Edmundson, A. B. Biophys. J. 1967, 7, 121. (9) Eisenberg, D.; Weiss, R. M.; Terwilliger, T. C. Nature 1982, 299, 371. (10) Segrest, J. P.; De Loof, H.; Dohlman, J. G.; Brouillette, C. G.; Anantharamaiah, G. M. Proteins 1990, 8, 103. (11) Finer-Moor, J.; Stroud, R. M. Proc. Narl. Acad. Sci. U.S.A. 1984, 81, 155. (12) Cronet, P.; Sander, C.; Vriend, G. Protein Eng. 1993, 6, 59. (13) Kerr, I. D.; Sansom, M. S . P. Eur. J. Biophys. 1993, 22, 269. (14) Brasseur, R. J. Biol. Chem. 1991, 266, 16120. (15) Furet, P.; Sele, A.; Cohen, N. C. J. Mol. Graphics 1988, 6, 182. (16) Efremov, R. G.; Gulyaev, D. I.; Vergoten, G.; Modyanov, N. N. J. Protein Chem. 1992, 11, 665. (17) Efremov, R. G.;Gulyaev, D. I.; Modyanov, N. N. J. Protein Chem. 1992, 11, 699. (18) Efremov, R. G.; Gulyaev, D. I.; Modyanov, N. N. J. Protein Chem. 1993, 12, 143. (19) Sansom, M. S . P.; Balaram, P.; Karle, I. L. Eur. Biophys. J. 1993, 21, 369. (20) Tirado-Rives, J.; Maxwell, D. S . ; Jorgensen, W. L. J. A m Chem SOC. 1993, 115, 11590. (21) Gerstein, M.; Lynden-Bell, R. M. J. Phys. Chem. 1993, 97, 2982. (22) Gerstein, M.; Lynden-Bell, R. M. J. Phys. Chem. 1993, 97, 2991. (23) Gerstein, M.; Lynden-Bell, R. M. J. Mol. Biol. 1993, 230, 641. (24) Bemstein, F. C.; Koetzle, T. F.; Williams, G. J. B.: Mever. E. F., Jr., Brice, M. D.; Rogers, J. R.; Kennard, 0.;Shimanouchi, T.; Tisumi, M. J. Mol. Biol. 1977, 112, 535. (25) Lemmon, M. A,; Flangan, J. M.; Treutlein, H. R.; Zhang, J.; Engelman, D. M. Biochemistry 1992, 31, 12719. (26) Canessa, C. M.; Horisberger, J.-D.; Rossier, B. Nature 1993, 361, 467. (27) Insight I1 User Guide,version 2.3.0; Biosym Technologies: San Diego, CA, 1993. (28) Jorgensen, W. L. BOSS 3.4, Yale University, New Haven, CT, 1993. (29) Jorgensen, W. L.; Tirado-Rives, J. J. Am. Chem. SOC. 1988, 110, 1657. (30) White, S. H.; Wimley, W. C. Curr. Opin. Struct. Biol. 1993,4,79. (31) Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K. J. Chem. In$ Comput. Sci. 1989, 29, 163. (32) Jorgensen, W. L. J. Phys. Chem. 1983, 87, 5304. (33) Henderson, R.; Baldwin, J. M.; Ceska, T. A.; Zemlin, F.; Beckmann, E.; Downing, K. H. J. Mol. Biol. 1990, 213, 899. (34) Deisenhofer, J.; Epp, 0.;Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618. (35) Treutlein, H.R.; Lemmon, M. A.; Engelman, D. M.; Brunger, A. T. Biochemistry 1992, 31, 12726. (36) Sansom, M. S. P. Prog. Biophys. Mol. Biol. 1991, 55, 139.
Acknowledgment. We thank Dr. W. L. Jorgensen for providing us with the BOSS program. R.G.E. was a Postdoctoral Fellow of the French Ministry of Research and an Associated Professor Fellow of the University of Science and Technology of Lille. The authors would like to thank the reviewers for their detailed and useful comments.
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