Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E

Martine Prévost1 and Jean-Pierre Kocher2

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structure and dynamics of the lipid-free LDL-receptor-binding domain of apolipoprotein E (apoE-RBD) has been investigated by Molecular Dynamics Simulations. ApoE-RBD in its monomeric lipid-free form is a singular four-helix bundle made up of four elongated amphipathic helices. Analysis of one 1.5 ns molecular dynamics trajectory of apoE-RBD performed in water indicates that the lipid-free domain adopts a structure that exhibits characteristics found in native proteins: it has very stable helices and presents a compact structure. Yet its interior exhibits a larger number of transient atomic-size cavities relative to that found in other proteins of similar size and its apolar side chains are more mobile. The latter features distinguish the elongated four-helix bundle as a slightly disordered structure, which shows a structural likeness with some de novo designed four-helix bundle proteins and shares with the latter a leucine-rich residue composition. We anticipate that these unique properties compared with other native helix bundles may be related to the postulated ability of apoE-RBD to undergo an opening of its bundle upon interaction with phospholipids. The distribution of empty cavities computed along the trajectory in the interface regions between the different pairs of helices reveals that the tertiary contacts in one of the interfaces are weaker suggesting that this particular interface could be more easily ruptured upon lipid association.

Keywords: apoE-RBD/LDL-receptor binding domain/four-helix bundle/Molecular Dynamics/protein packing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apolipoprotein E (apoE), a blood plasma protein, serves to regulate lipoprotein metabolism and to control the transport and redistribution of lipids among tissues and cells. It also binds to cell surface lipoprotein receptors and thereby mediates the cellular uptake of most lipoproteins (for a review, see Weisgraber, 1994). As a result of its widespread presence in amyloid plaques, it has been suggested that apoE may also function as a pathological chaperone protein, that is, one that induces ß-pleated conformation in amyloidogenic polypeptides (Wisniewski and Frangione, 1992Go). ApoE contains two regions that fold independently and differ markedly in their stability. Unlike the C-terminal fragment, the N-terminal domain is a highly stable monomer featuring a stability in the range of soluble proteins. The two structural domains have different functional properties, the N-terminal domain contains the receptor-binding function and the C-terminal domain controls the lipid-binding function.

The structure of the N-terminal domain or LDL-receptor binding domain (apoE-RBD) was solved by X-ray diffraction studies (Wilson et al., 1991Go). Its structure known at 2.25 Å resolution contains five helices, four of which are arranged in an antiparallel four-helix bundle (Figure 1Go). 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., 1991Go). 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., 1992Go).



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Fig. 1. Tube diagram of one snapshot structure of the LDL-receptor domain of apolipoprotein E (apoE-RBD). The backbone of the four helices (H1–H4) forming the bundle as well as the short helix connecting helix 1 and 2 (H2') is drawn in dark grey. Loops connecting the helices are shown in white. Residues comprising helices and the longest loop are given in parentheses. Locations in the protein matrix (including the protein–water interface) which accommodate 1.4 Å cavities (light grey dots) occurring in the first 300 ps portion of the apoE-RBD 1.5 ns trajectory. The structures were superimposed on the first structure generated in the 300 ps trajectory using C{alpha} atoms.

 
Albeit the binding of apoE to lipoprotein particles appears to be mediated by the C-terminal domain (residues 219–299), the LDL-receptor binding domain (residues 1–166) does associate in vitro with phospholipids to form discoidal particles. Furthermore, it is known that lipid association is required for high-affinity binding of the N-terminal domain of apoE to the LDL receptor. As the hydrophobic side chains, which are directed towards the interior of the bundle, are thought to interact with the hydrophobic acyl chains of the phospholipids, it has been postulated that lipid binding of apoE-RBD may involve a major conformational change whereby the hydrophobic face of one or more helices becomes accessible for interaction with non-polar phospholipid tails. This model, in which the bundle opens without a major disruption of the {alpha}-helical structure, is supported by the surface properties measured at the air–water interface (Weisgraber, 1994Go). Recent spectroscopic studies (Raussens et al., 1996Go; Wang et al., 1997Go) have proposed that apolipophorin III, an apolipoprotein structurally similar to apoE-RBD, undergoes the same large conformational change upon lipid binding. One of these studies (Raussens et al., 1996Go) observed an exchange rate of amide protons higher in the lipid-associated form than in the lipid-free form of the protein, which may originate from alterations in the tertiary structure of the former form. Furthermore, the rate of H/D exchange of the lipid-free protein hints that its interior could not be as ordered as that in other native proteins. The putative conformational change is also supported by experiments showing that membrane and phospholipid vesicles can conduct large conformational changes in proteins, transforming them into a much more flexible state (de Jongh, 1992; Pinheiro et al., 1997Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular dynamics simulations

The simulation was carried out starting with the high-resolution crystallographic coordinates of apoE-RBD (Wilson et al., 1991Go) using the program CHARMM (Brooks et al., 1983Go). 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., 1983Go). Bonds connecting hydrogens were constrained using the SHAKE algorithm (Ryckaert et al., 1977Go) 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., 1990Go). 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 protein–protein and protein–solvent interactions is correct (Prévost, 1998Go).

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 {Delta}Ac of cavity formation was computed from the likelihood of encountering empty cavities of a given size in thermally equilibrated molecular systems (Lee, 1985Go) using a procedure previously implemented in simulations of pure liquids (Pohorille and Pratt, 1990Go; Prévost et al., 1996Go) and recently applied to proteins (Kocher et al., 1996Go). {Delta}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, 1983Go) 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., 1996Go).

Hydrogen bonds were identified using the molecular modeling package BRUGEL (Delhaise et al., 1985Go) with the following criteria: a hydrogen-acceptor distance must be <=2.7 Å, the donor–hydrogen–acceptor and the hydrogen–acceptor–`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, 1991Go).

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We report here an analysis of global as well as specific properties of apoE-RBD in one 1.5 ns trajectory in order to get greater insight into the dynamics and structure of an elongated four-helix bundle, to inspect the compactness of the tertiary structure and to monitor the steadiness of the lengthy helices. We also report a comparative analysis of the crystal structure of apoE-RBD with those of native and de novo designed four-helix bundles.

Tertiary structure

R.m.s. deviation The root mean square deviation (r.m.s.d.) of C{alpha} atoms of individual conformations of the apoE-RBD fragment was computed along the 1.5 ns trajectory (Figure 2Go). 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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Fig. 2. R.m.s.d. of the C{alpha} atoms computed from the crystal structure as a function of the simulation time. Values correspond to snapshot structures separated by 0.2 ps along the 1.5 ns trajectory.

 
Accessible surface area of side chains The solvent-accessible surface area of the side chains that belong to the helices forming the bundle was computed in every conformation, averaged over the 1.5 ns trajectory and normalized by a standard value of the corresponding residue computed in an extended tripeptide (Figure 3Go). A 3–4 periodicity in the solvent-accessible area of the side chains is manifested along the four helices. Side chains presenting a zero-accessibility are chiefly hydrophobic and in a few cases aromatic. Helices 1 and 4 exhibit a clear periodicity that is never contradicted. However in helices 2 and 3, some residues which would be expected to be buried given the 3–4 periodicity are not (residues 64, 74–75 in helix 2 and residues 90 and 93 in helix 3). Noticeably, while helices 1, 2 and 3 present a 7- (approximately two helix turns) or 11-repeat (approximately three helix turns) of buried leucine amino acids, helix 4 respects a strict 4-repeat (approximately one helix turn) of the burial of this amino acid.






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Fig. 3. Side chain dihedral angle fluctuations ({Delta}{chi}1, {Delta}{chi}2), normalized solvent-accessible surface area of the side chains (SASA) and ii+4 H bond distances averaged over the 1.5 ns trajectory as a function of the residue number in (a) helix 1 (residues 24–42), (b) helix 2 (residues 54–81), (c) helix 3 (residues 87–125) and (d) helix 4 (residues 130–164). Closed circles indicate sequestered residues as well as their corresponding ii+4 H bond distance (see text).

 
Cavity formation in the protein matrix The free energy of cavity formation, {Delta}Ac as a function of the cavity size can be used as a probe of the physical properties of the protein matrix (Kocher et al., 1996Go). In particular, it is used in this study to investigate the possible looseness of the protein interior and to locate where large cavities tend to occur. The free energy of cavity formation is computed from the likelihood of finding a cavity of a given size in the thermally equilibrated systems: for vanishingly small cavity sizes, {Delta}Ac(Rc = 0) is related to the packing density. For larger cavity sizes, {Delta}Ac reveals how the empty space is distributed throughout the medium.

It has been shown (Kocher et al., 1996Go) that {Delta}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, {Delta}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 4Go). The five {Delta}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 {Delta}Ac value of about 8 kcal/mol.



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Fig. 4. Size-dependence of the free energy of cavity formation {Delta}Ac in buried regions of apoE-RBD. The five displayed curves plotted as a function of the cavity radius Rc correspond to values computed from five consecutive 300 ps portions of the 1.5 ns trajectory. The {Delta}Ac curves vary insignificantly with the different 300 ps portions except for cavity sizes >=1.5 Å.

 
Figure 5Go displays {Delta}Ac values as a function of the cavity radius Rc, computed in 300 ps MD trajectories of three proteins: barnase, T4 phage lysozyme and apoE-RDB. Packing density, obtained from {Delta}Ac (Rc = 0), is rather similar for the three proteins: 0.66 for barnase and 0.65 for T4 lysozyme and apoE-RBD. However, {Delta}Ac for Rc > 0.5 Å is lower in apoE-RBD than in either of the other two proteins. Thus apoE-RBD, which is about the same size as barnase and T4 lysozyme, harbors a larger number of cavities of large size, lending reason to the belief that apoE-RBD features a looser compactness than the other two proteins.



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Fig. 5. Cavity size-dependence of the free energy of cavity formation {Delta}Ac in apoE-RBD, barnase and T4 phage lysozyme (T4 lyso). The three displayed curves plotted as a function of the cavity radius Rc correspond to values computed in buried and accessible protein regions from 300 ps trajectories.

 
Packing density computed in buried regions of these three proteins is lower in apoE-RBD, in T4 lysozyme and its domain 2 (0.67, 0.67 and 0.66 respectively) than in barnase and domain 1 of T4 lysozyme (0.7 and 0.71 respectively). The cost of forming cavities over the entire range of cavity sizes is lower in the apoE-RBD interior than in barnase and in domain 1 of the T4 lysozyme interior and similar to that in T4 lysozyme and its domain 2 (data not shown). It was previously suggested that the large localized permanent cavities distinguishing domain 2 of T4 lysozyme explain the lower cost in forming atomic-size cavities relative to that in barnase (Kocher et al., 1996Go). As apoE-RDB does not feature any large permanent defect, as is the case for domain 2 of T4 lysozyme, its lower cost in forming cavities can only be interpreted as an increased occurrence of transient atomic-size cavities.

Cavities are not uniformly distributed in apoE-RBD but are concentrated in a few circumscribed regions (Figure 1Go). 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 6Go).





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Fig. 6. Locations in three buried regions of apoE-RBD which accommodate 1.4 Å cavities (dots) in a 300 ps portion of the 1.5 ns trajectory. The backbone of the first generated conformation in the 300 ps trajectory of apoE-RBD is indicated as a tube. (The other conformations were superimposed on the first structure using C{alpha} atoms.) Side chains enclosing the cavities are depicted as sticks. These buried regions are located along the four-helix-bundle axis: (a) residues from helices 2, 3 and 4 that are close to the loop (82–86) connecting helix 2 and 3; (b) leucine, methionine as well as one threonine and one tryptophan side chain protruding from the four helices of the bundle; (c) leucine side chains as well as the hydrophobic portion of a lysine side chain from helices 2, 3 and 4.

 
There are two ways in which the protein can spread on the surface upon lipid-binding: (i) by the movement of helices 1 and 2 in one direction and helices 3 and 4 in the other direction around a hinge involving the loop between helices 2 and 3 (residues 82–86); or (ii) by the movement of helices 1 and 4 in one direction and helices 2 and 3 in the other around two hinges, one is the small helix 2' (residues 45–52) and the other the loop between helices 2 and 3 (residues 126–129). In each pathway, one of the two interfaces between pairs of helices would be ruptured. Figure 7Go shows that for all five consecutive trajectories of 300 ps, cavities smaller than 1 Å radius occur more frequently at the interface between the pair of helices 1 and 2 (H1–H2) and the pair of helices 3 and 4 (H3–H4) than at the interface between the pairs of helices H1–H4/H2–H3. Likewise, the packing density computed from {Delta}Ac(Rc = 0) in the former interface is lower relative to that in the latter by about 10%. The average of the contact area, computed for every conformation in the 1.5 ns trajectory, between the pairs H1–H2/H3–H4 is 30% lower than that between the pairs H1–H4/H2–H3. This observation is, however, not unexpected as helices 3 and 4 are the largest in the bundle and taking them apart will imply the breaking of a larger interface area. The contribution of the hydrophobic contact area is 75% and that of charged residues 9% for the H1–H4/H2–H3 interface, while those figures drop to 70 and 5% respectively for the H1–H2/H3–H4 interface.



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Fig. 7. Cavity size-dependence of the free energy of cavity formation {Delta}Ac in the buried interfaces formed between pairs of the helices forming the bundle. The displayed curves plotted as a function of the cavity radius Rc correspond to values computed for five consecutive 300 ps trajectories in the 1.5 ns trajectory (solid line) in the buried interface between the pair of helices 1 and 2 (H1–H2) and that of helices 3 and 4 (H3–H4) and (dashed line) in the buried interface between the pair of helices 1 and 4 (H1–H4) and that of helices 2 and 3 (H2–H3).

 
Side chain mobility The computed average fluctuation of the side chain torsion angles {chi}1 and {chi}2, which are the most constrained angles due to their nearness to the main chain, for the residues that compose the bundle, provides supplementary pieces of information for investigation of the degree of packing in the protein interior. Fluctuations in {chi}1 and {chi}2 angles higher than 25° (Figure 3Go) are frequently observed even for fully buried side chains; for instance, in the case of Leu60 and Met64 in helix 2; Leu97, Gln101 and Val122 in helix 3; and Leu133, Leu141, Leu144, Leu148, Leu155 and Leu159 in helix 4. These values are close to those observed for {chi}2, {chi}3 (the latter being further away from the main chain than {chi}1, {chi}2 and thus expected to be less constrained) in a simulation of the apo form of cytochrome b562, suspected to behave as a molten globule (Laidig and Daggett, 1996Go). Furthermore, {chi}1 and {chi}2 angles in apoE-RBD have larger fluctuations than those computed in a 500 ps trajectory of native barnase, in which an overwhelming majority of side chains belonging to secondary structures exhibit {chi}1, {chi}2 fluctuations not larger than 20° (M.Prévost, unpublished results), and in trajectories of other simulated proteins (Laidig and Daggett, 1996Go). Hence apoE-RBD, as the apo form of cytochrome b562, evidences a remarkable flexibility of its side chains inferring that side chain packing is not arranged as in a lock-and-key fit.

Secondary structures

H bonds in secondary structures The behaviour of backbone H bonds formed within the bundle is depicted in Figure 8Go. 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 29–33 in helix 1; 72–77 in helix 2; 102–107 and 110–121 in helix 3; and 146–150 in helix 4. Most of the backbone atoms whose side chains are deeply buried within the four-helix bundle (Figure 3Go) 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 {alpha}-helical segments in protein crystal structures (Sundaralingam and Sekharudu, 1989Go).



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Fig. 8. Density plot for the relative population of intra-molecular backbone H bonds formed within the helices of the bundle averaged over the 1.5 ns trajectory of apoE-RBD. The squares are shaded according the magnitude of the population. Only H bonds featuring a population higher than 25% have been retained.

 
Distances for the ii+4 H bonds in the helices of apoE-RBD were computed and averaged over the 1.5 ns trajectory (Figure 3Go). H bond distances change periodically along the helices with a 3–4 repeat pattern: H bonds are shorter in the hydrophobic face and longer on the solvent-exposed face. However, this observation needs to be refined: helix 1 manifests a clear periodicity of the H-bond distances, the shortest of which corresponds to buried hydrophobic and aromatic side chains. In helix 2, the periodicity is less manifest. However, the amino acid sequence corresponding to the buried residues is not purely hydrophobic as some residues are either polar (Thr67) or even charged (Lys75). In helix 3, the periodicity is attenuated in the first two turns, which do not exhibit an entirely buried face, and perturbed towards the center of the helix, possibly due to the presence of a polar buried residue (Gln101) and of Gly105 inducing a kink in the helix. Helix 4, in contrast, exhibits a striking 3–4 periodicity in the H bond distance. The amino acid sequence in its hydrophobic face is a nearly uninterrupted succession of leucine residues punctuated by one alanine (152) and one tyrosine (162).

Backbone dihedral angles The backbone dihedral angles ({Phi},{Psi}) were monitored as an additional probe of secondary structure content. The ({Phi},{Psi}) values of the residues belonging to the five helices as defined in the crystal structure (see Figure 1Go 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, 1996Go). Likewise the {alpha}4 peptide, a leucine-rich peptide that forms an {alpha}-helical tetramer, adopts a structure with properties intermediate between those of the native and molten globule states of proteins (Handel et al., 1993Go). To induce a more native-like state, two metal chelating histidine residues were introduced at leucine positions (Handel et al., 1993Go). 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.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
H bonds and backbone dihedral angles are important landmarks of protein conformations. A majority of backbone H bonds formed in the helices of the crystal structure persist in the 1.5 ns trajectory indicating, in agreement with the average values of {Phi},{Psi} angles, that the helices are rather stable as they form in the crystal structure. This observation may sound commonplace as far as simulations of typical four-helix bundles are concerned yet to our knowledge it is shown for the first time for such elongated helices.

An evident correlation is found between the location of the residue on the helix and the ii+4 hydrogen bond length (Figure 3Go). 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 {alpha}-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 3–4 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 3Go) and the loss of the 3–4 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., 1992Go) in which it was shown that H bonds in an isolated amphipathic {alpha}-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 {alpha}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., 1993Go). 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 H1–H2/H3–H4 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 82–86). 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 H1–H2/H3–H4 (Nagi and Regan, 1996Go). The observation that loop 82–86 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., 1991Go). 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. H2–H3/H1–H4 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., 1997Go) 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., 1996Go; Uversky et al., 1997Go) 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., 1997Go) 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.


    Acknowledgments
 
The authors are grateful to B.K.Lee and D.Van Belle for helpful discussions and critical reading of the manuscript and to Joe Camissa for his invaluable help with the plots. The authors thank UCMB (Université Libre de Bruxelles) for access to its computers. M.P. is a research associate at the National Fund for Scientific Research (Belgium). J.-P.K. is a visiting fellow at the National Institutes of Health, Bethesda, MD, USA. All molecular graphics images were produced using the program GRASP (Nicholls et al., 1991Go).


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 16, 1998; revised January 20, 1999; accepted March 3, 1999.





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