Ingénierie Biomoléculaire, Université Libre de Bruxelles, CP 165/64,Av. F. Roosevelt, B-1050 Bruxelles, Belgium and 2 Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Abstract |
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Keywords: apoE-RBD/LDL-receptor binding domain/four-helix bundle/Molecular Dynamics/protein packing
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Introduction |
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The structure of the N-terminal domain or LDL-receptor binding domain (apoE-RBD) was solved by X-ray diffraction studies (Wilson et al., 1991). Its structure known at 2.25 Å resolution contains five helices, four of which are arranged in an antiparallel four-helix bundle (Figure 1
). Loops connect the helices to each other with the exception of the link between helix 1 and 2 which is a short helix. The structure of apoE-RBD is unusual among four-helix bundle proteins in that three of the four helices are much longer (28, 36 and 35 residues) than the average helical length (18 residues), although apolipophorin III, another apolipoprotein, also contains an elongated helical bundle (Breiter et al., 1991
). The helices in apoE-RBD are amphipathic in that the hydrophobic side chains are sequestered in the interior of the bundle and the hydrophilic side chains are solvent-exposed on the surface. The arrangement of leucine side chains, which occur approximately every seven residues, forms a leucine zipper-like structure (Segrest et al., 1992
).
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Even though there is a large body of structural information on apoE-RBD, precise characterization of the flexibility of the protein interior and of the factors likely to promote the bundle opening is difficult as these processes are dynamic by nature. A few questions can be thus addressed with molecular dynamics (MD) simulations of apoE-RBD in aqueous solution and in a water/lipid mixture. How packed is the protein interior? How much flexibility do the side chains sequestered into the protein core experience? Are the secondary structures of such an elongated four-helix bundle stable? The answers to these questions may deliver a dynamic picture of the molecule, complementary to the picture witnessed by experimental data.
To this end, a MD study of apoE-RBD in water at room temperature was performed for 1.5 ns. To our knowledge, this is the first simulation of such an elongated four-helix bundle. ApoE-RBD is not a large molecule; its crystal structure contains 144 amino acid residues. The analysis of the trajectory suggests a structural model in which the protein interior packing is somewhat looser than that of other native proteins attested by an increased flexibility of the side chains and a concomitant weakness of tertiary contacts as witnessed by the occurrence of transient atomic-size cavities. The lengthy helices remain well shaped and thus do not seem to contribute to the perceived disorder of the protein interior. Interestingly, the structural analysis of other native and de novo designed four-helix bundles shows that apoE-RBD shares with the latter a loose packing as well as a leucine-rich residue composition.
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Materials and methods |
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The simulation was carried out starting with the high-resolution crystallographic coordinates of apoE-RBD (Wilson et al., 1991) using the program CHARMM (Brooks et al., 1983
). The protein and solvent interacted via the CHARMM 22 force field where all protein atoms are explicitly represented (McKerell et al., 1998) and where the water is represented by the TIP3P model (Jorgensen et al., 1983
). Bonds connecting hydrogens were constrained using the SHAKE algorithm (Ryckaert et al., 1977
) which permitted the use of an integration time step of 1 fs. The simulation consisted of a system defined by the protein plus 5535 water molecules in a periodic volume with dimensions (62.1/46.575/65.205 Å). The water density in the simulation box is 0.992 g/ml.
Long-range interactions were smoothly truncated at 8.5 Å with a shifting function for the electrostatic interaction and a switching function for the van der Waals interaction, the latter being applied between 7.5 and 8.5 Å. The truncation scheme applied to the calculation of electrostatic interactions was calibrated against the Ewald summation method in simulations of pure liquid water and was shown to perform best with respect to both structural and thermodynamic properties (Prévost et al., 1990). Moreover, an analysis of the 1.5 ns trajectory, so as to monitor the burial of water molecules within the protein interior, revealed that one water molecule buried in the crystal structure exchanges in the course of the simulation with one water molecule initially located in the bulk water, indicating that the balance of proteinprotein and proteinsolvent interactions is correct (Prévost, 1998
).
After 35 ps equilibration of the water structure in the presence of the fixed protein, the system was slowly brought up to a temperature of 300 K for 40 ps and equilibrated for 120 ps, after which 1.5 ns of production run was carried out in the microcanonical ensemble. Analysis was performed on conformations collected every 0.2 ps.
Computing the free energy cost of cavity formation
The Helmoltz free energy Ac of cavity formation was computed from the likelihood of encountering empty cavities of a given size in thermally equilibrated molecular systems (Lee, 1985
) using a procedure previously implemented in simulations of pure liquids (Pohorille and Pratt, 1990
; Prévost et al., 1996
) and recently applied to proteins (Kocher et al., 1996
).
Ac values were computed using the modeling package CASSIO (Kocher,J.-P., personal communication).
Analysis of structural properties
Secondary structures in the crystal conformation were defined by using the DSSP criteria (Kabsch and Sander, 1983) and extending the fragment at both ends as long as the backbone dihedral angle values remain in domains comprising either all right-handed conformations or all extended conformations (Wintjens et al., 1996
).
Hydrogen bonds were identified using the molecular modeling package BRUGEL (Delhaise et al., 1985) with the following criteria: a hydrogen-acceptor distance must be
2.7 Å, the donorhydrogenacceptor and the hydrogenacceptor`from' angles must be
90° (`from' stands for the atom covalently linked to the acceptor atom).
The contact area between pairs of helices was computed as the sum of the area of the polyhedra faces that atoms of a given residue pair have in common. The polyhedra calculation was performed using a method based on radical planes implemented in the program SurVol (Alard, 1991).
The interhelical spacing between two helices was defined as the distance between the helical geometric centers computed as the average of the backbone atom positions.
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Results |
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Tertiary structure
R.m.s. deviation
The root mean square deviation (r.m.s.d.) of C atoms of individual conformations of the apoE-RBD fragment was computed along the 1.5 ns trajectory (Figure 2
). The r.m.s.d., measured relative to the starting crystal conformation, displays a value of about 1 Å at the beginning of the trajectory (t = 0), indicating that movements have occurred during the thermalization and equilibration periods (120 ps) that precede the production run. It then fluctuates roughly between 1 and 1.5 Å during the 1.5 ns trajectory.
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It has been shown (Kocher et al., 1996) that
Ac values differ in different proteins and in different domains of the same protein. It is thus important at this point to discuss the accuracy of the computed values. The estimated statistical error is negligible for very small cavities and reaches at most 0.05 kcal/mol for cavities of 1.5 Å. However, there could be a systematic error arising from insufficient sampling of conformational space. In order to verify that our calculations have achieved adequate sampling of conformational space during the 1.5 ns simulation time,
Ac values were computed in buried regions of the protein in five successive trajectories of 300 ps length each in the 1.5 ns trajectory (Figure 4
). The five
Ac curves show small differences, except for large cavity sizes (
1.5 Å). The largest difference being observed for a cavity size of 1.8 Å is 0.8 kcal/mol relative to a
Ac value of about 8 kcal/mol.
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Cavities are not uniformly distributed in apoE-RBD but are concentrated in a few circumscribed regions (Figure 1). A detailed inspection of the buried regions clearly indicates that cavities of 1.4 Å (1.4 is about the size of a water molecule) occur preferentially in locations where hydrophobic side chains pertaining to the helices of the bundle meet each other (Figure 6
).
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Secondary structures
H bonds in secondary structures
The behaviour of backbone H bonds formed within the bundle is depicted in Figure 8. Almost all crystal H bonds, mainly ii+4 and a few ii+3 H bonds, persist during the 1.5 ns simulation though fluctuantly. The ii+3 native H bonds are more transient than the ii+4 ones. On the whole, H bonds involving residues of the N- and C-termini lack the persistence of H bonds formed by backbone atoms further away from the helix boundaries. Among the latter a few manifest in each helix a transience; for example, H bonds involving residues 2933 in helix 1; 7277 in helix 2; 102107 and 110121 in helix 3; and 146150 in helix 4. Most of the backbone atoms whose side chains are deeply buried within the four-helix bundle (Figure 3
) are engaged in strongly persistent backbone H bonds. A rather large proportion of the backbone carbonyl groups in the helices of the bundle, in particular the carbonyl groups that are not fully buried and those that are not engaged in bifurcated ii+4 and ii+3 H bonds, make additional H bonds with water molecules. The pattern of H bonds observed is that of a backbone carbonyl group H bonded to its backbone amide group partner and to an external water molecule. A similar pattern was pinpointed in a detailed analysis of
-helical segments in protein crystal structures (Sundaralingam and Sekharudu, 1989
).
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Backbone dihedral angles
The backbone dihedral angles (,
) were monitored as an additional probe of secondary structure content. The (
,
) values of the residues belonging to the five helices as defined in the crystal structure (see Figure 1
for definitions) averaged over the conformations generated in the 1.5 ns trajectory fall in the helical region (data not shown). The few that do not are either N- or C-terminal residues of the helices.
Structural characteristics of apoE-RBD versus those of native and de novo four-helix bundles Comparison between interhelical spacings in apoE-RBD structure and those featuring other four-helix bundles indicates a large variety of values and thus raises the general question of how the side chains interlock in a bundle interior. To shed light on this issue we inspected three crystal structures featuring a four-helix bundle fold: cytochrome b562, ferritin and myohemerythrin and compared them with apoE-RBD in terms of residue composition and packing arrangement of the interior side chains. Compared with cytochrome b562, myohemerythrin and ferritin, the residue composition of the apolipoprotein interior is more homogeneous. Its core is chiefly hydrophobic; only a few interactions involving fully buried polar or charged side chains were observed. The protein interior is overwhelmingly composed of leucine residues, flecked with a few valines, alanines and aromatic residues. The interhelical spacings in apoE-RBD is 10.2 Å between adjacent helices and 15 Å between diagonal pairs of helices. The interhelical spacings in the cytochrome b562 crystal structure are shorter than those observed in apoE-RBD. In the layers of side chains forming the cytochrome b562 core, the buried residues consist essentially of an almost equal number of mingling alanine and leucine amino acids, suggesting that alanine shorter side chains would favour a closer approach of the helices. Myohemerythrin like apoE-RBD exhibits quite large interhelical spacings. The inspection of the core-forming residues reveals a cluster of five to six histidines enclosing deeply buried glutamic acid and aspartic acid side chains, giving rise to specifically oriented interactions and to large interhelical spacings. The composition of the other centrally buried residues is hydrophobic and quite heterogeneous with leucine and isoleucine amino acids. Ferritin, for which interhelical spacing values are closer to those in cytochrome b562, has also a heterogeneous core formed by the interdigitation of various types of hydrophobic residues: leucine and alanine as well as a few aromatic residues such as phenylalanine or tyrosine. However, polar residues (Thr, Ser, Gln) are almost as frequent as hydrophobic amino acids and contribute by H bonding to the packing of the bundle core.
It has been previously shown that the unmixed presence of leucine amino acids in the interior of four-helix bundle structures induces molten globule characteristics. A helical tetramer whose interior is filled with leucine side chains was shown to exhibit the characteristics of a molten globule whereas its modified sequence with valine replacing leucine at buried key positions led to a more native-like behaviour (Betz and DeGrado, 1996). Likewise the
4 peptide, a leucine-rich peptide that forms an
-helical tetramer, adopts a structure with properties intermediate between those of the native and molten globule states of proteins (Handel et al., 1993
). To induce a more native-like state, two metal chelating histidine residues were introduced at leucine positions (Handel et al., 1993
). These studies on de novo designed tetramer-associating peptides suggested that leucine residues at buried key positions are sufficient to drive the polypeptide chain to fold into helical secondary structures and to assemble into a bundle, but that a more native-like structure required more specific interactions involving polar residues or at least a larger heterogeneity of the buried hydrophobic amino acids.
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Discussion |
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An evident correlation is found between the location of the residue on the helix and the ii+4 hydrogen bond length (Figure 3). Our simulation shows that H bonds in the four helices are shorter on the buried hydrophobic face and longer on the solvent-exposed faces. As no distortion of the
-helical structure itself occurs this periodic repeat is likely to be due to local distortions in the vicinity of the residues engaged in backbone hydrogen bonding. A plot of hydrogen bond lengths versus amino acid sequence for helices 2 and 3 does not show a 34 repeat periodicity as salient as that for helices 1 and 4. Noticeably helices 1 and 4 exhibit a higher hydrophobicity of their hydrophobic face (Figure 3
) and the loss of the 34 repeat periodicity seems to arise when the residue located on the hydrophobic face or its direct environment within the helix displays a decrease in hydrophobicity. These results are in agreement with an NMR study (Zhou et al., 1992
) in which it was shown that H bonds in an isolated amphipathic
-helical peptide are shorter in the center of the hydrophobic face and longer in the center of the hydrophilic face. Likewise the helical peptide was shown to exhibit a curvature whose degree depends on the extent of helix amphipathicity and which was suggested to result in the side chains in the center of the hydrophobic face forming a hydrophobic microenvironment.
ApoE-RBD manifests a duality in its behaviour in aqueous solution. On one hand, the free-lipid form manifests well-known characteristics of native proteins: low r.m.s.d. and stable secondary structures. On the other hand, it exhibits a rather high occurrence of transient atomic-size cavities and a large side chain flexibility in its interior that makes it a slightly disordered structure differing from the interior of native states of proteins and showing similarities with molten globule states. The comparison with other native four-helix bundle structures suggests that apoE-RBD lacks the more heterogeneous residue composition that permits the folding into a tightly packed structure. ApoE-RBD also manifests some characteristics of de novo designed four-helix bundle proteins: rich in leucine and featuring a high stability, but whose tertiary structure is marked by a weakness of tertiary compactness. It might appear as paradoxical that apoE-RBD, which has a thermodynamic stability as high as that of a globular protein, would behave as a slightly disordered state. This dual characteristic has, however, been observed for the leucine-rich 4 peptide that folds into a protein with extreme stability, but also with a conformation that is less structured than the native state of most proteins (Handel et al., 1993
). To rationalize this observation, the authors suggested that the flexibility of the leucine side chain, which has a large number of low-energy rotamers, contributes to a large stabilizing entropy. This view could be applied to apoE-RBD as a few buried leucine side chains exhibit a rather large flexibility.
Whether the features of apoE-RBD interior would serve to promote the conformational change induced upon lipid binding or is a characteristics of such an elongated bundle, that, with its residue composition, could not form a structure as packed as other proteins, remains however to be established.
The observation that the hydrophobic contact area in the H1H2/H3H4 interface is smaller, together with the finding that it is easier to grow cavities within this interface infers that the latter would be easier to fracture in aqueous solutions as less hydrophobic contacts would be lost. An additional argument to advocate this pathway is that the latter involves a movement about the longest loop of the protein (residues 8286). From an entropic standpoint the closure of longer loops should require more energy and thus facilitate protein destabilization against the favourable interaction between the pairs of helices H1H2/H3H4 (Nagi and Regan, 1996). The observation that loop 8286 in the apoE-RBD crystal structure is ill-defined makes the assertion of its role in the lipid-binding-induced conformational change difficult (Wilson et al., 1991
). However, this lack of a well-defined structure may originate from high flexibility and thus sustains the view that a hinge-type opening about this loop would be likely to occur. One would, however, expect an increase in the hydrophobicity of the hydrophobic face of the apolipoprotein to favour the lipid affinity (Anantharamaiah, 1986) and one would hence predict, in contrast to the former arguments, that it would be more favourable to bury the largest hydrophobic interface, i.e. H2H3/H1H4 upon interaction with the lipids.
All this reasoning, however, does not take into account the effect of the lipid environment on the triggering of the conformational transition. A study of cytochrome c in the presence of phospholipid vesicles (Pinheiro et al., 1997) emphasizes a mechanism for lipid-induced conformational change triggered by a local decrease in effective pH, while another performed on cytochrome c in organic solvents (Bychkova et al., 1996
; Uversky et al., 1997
) proposes that the conformational transitions would be induced by a decrease in dielectric constant near the membrane-like interface. A study on apolipophorin-III (Wang et al., 1997
) suggests that the short helix connecting two helices of the bundle, structurally similar to helix 2' in apoE-RBD, could play an important role in terms of initiating interactions with lipoprotein surfaces. Further computer experiments are currently being performed to study at the microscopic level the possible interactions of apoE-RBD with a mimicking-bilayer environment.
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Acknowledgments |
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Notes |
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References |
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Received September 16, 1998; revised January 20, 1999; accepted March 3, 1999.