Molecular Determinants of the Interaction ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

Molecular Determinants of the Interaction Between the CTerminal Domain of Alzheimer’s ␤-Amyloid Peptide and Apolipoprotein E ␣-Helices Laurence Lins, Annick Thomas-Soumarmon, *Thierry Pillot, †Joe¨l Vandekerckhove, *Maryvonne Rosseneu, and ‡Robert Brasseur INSERM U. 10, Hoˆpital Bichat-Claude Bernard, Paris, France; *Laboratory for Lipoprotein Chemistry and †Department of Biochemistry, Flanders Interuniversity Institute for Biotechnology, University Gent, Gent; and ‡Centre de Biophysique Mole´culaire Nume´rique, Faculte´ des Sciences Agronomiques de Gembloux, Gembloux, Belgium

Abstract: In a previous work, we predicted and demonstrated that the 29 – 42-residue fragment of ␤-amyloid peptide (A␤ peptide) has in vitro capacities close to those of the tilted fragment of viral fusion proteins. We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of A␤(29 – 42) via a direct interaction. Therefore, we suggested that this fragment is implicated in the neurotoxicity of A␤ and in the protective effects of apolipoprotein E in Alzheimer’s disease. Because structurally related apolipoproteins do not interact with the A␤ C-terminal domain but inhibit viral fusion, we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion. In this study, we simulated interactions of all amphipathic helices of apolipoproteins E and A-I with A␤ and simian immunodeficiency virus (SIV) fusogenic fragments by molecular modeling. We further calculated cross-interactions that do not inhibit fusion in vitro. The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of A␤(29 – 42) and SIV peptides. Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C-terminal amphipathic helices. Key Words: ␤-Amyloid peptide — Apolipoprotein E—Tilted peptide —Molecular modeling— Hydrophobicity. J. Neurochem. 73, 758 –769 (1999).

APP cleavage, as it is found in human CSF (Seubert et al., 1992; Shoji et al., 1992) and in the conditioned medium of a wide variety of cell cultures (Haass et al., 1992; Busciglio et al., 1993). In contrast, A␤(1– 42) is the predominant species in amyloid plaques (Iwatsubo et al., 1994). Although the presence of A␤ deposits in brain is a major pathological hallmark of AD, controversy exists about the central role of A␤ peptide in the pathological development. The “amyloid” theory is supported by two main lines of evidence (Yankner, 1996): the association of A␤ peptide with degenerating neurons in the brain of AD patients and the link between mutations in APP and inherited forms of AD. Those AD-inducing mutations result in an increased production of A␤ peptide (Cai et al., 1993; Suzuki et al., 1994; Mann et al., 1996). Furthermore, it was recently shown that the presenilin mutations associated with AD development result in increased A␤(1– 42) production (Selkoe, 1997). Recent studies suggest that A␤ has neurotoxic properties, notably in the aggregated state (Busciglio et al., 1993; Pike et al., 1993; Mattson, 1995). Several reports have demonstrated that the C-terminal domain of A␤ is critical for amyloid aggregation and fibril stabilization (Halverson et al., 1990; Lansbury et al., 1995). Endogenous factors affect the rate of amyloid formation and could play a significant role in the pathogenesis of AD. Recent epidemiologic and biochemical observations suggest that apolipoprotein E (apo E), a key apo-

The occurrence of Alzheimer’s disease (AD) is associated with brain vascular and neuronal damage involving the formation of intracellular neurofibrillar tangles and the accumulation of the ␤-amyloid peptide (A␤ peptide) in extracellular plaques (Selkoe, 1991, 1994; Kosik, 1992; Sisodia and Price, 1995). A␤, 39 – 42 residues long, is synthesized in glial cells and in neurons as part of a much larger transmembrane protein called ␤-amyloid precursor protein (APP) (Sisodia and Price, 1995). The A␤(1– 40) peptide is a normal product of

Received November 20, 1998; revised manuscript received April 16, 1999; accepted April 16, 1999. Address correspondence and reprint requests to Dr. L. Lins at INSERM U. 10, Hoˆpital Bichat-Claude Bernard, 170, Boulevard Ney, F-75018 Paris, France. Abbreviations used: A␤, ␤-amyloid peptide; AD, Alzheimer’s disease; apo, apolipoprotein; APP, ␤-amyloid precursor protein; MHP, molecular hydrophobicity potential; MLP, molecular lipophilic potential; SIV, simian immunodeficiency virus.

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A␤–APOLIPOPROTEIN E INTERACTION lipoprotein in lipid metabolism, plays such a role in brain (Weisgraber et al., 1994; Wisniewski et al., 1995). Three common variants of apo E are found in the human population, namely, the ⑀2, ⑀3, and ⑀4 alleles, corresponding, respectively, to the apo E2, E3, and E4 isoforms. They differ by a single amino acid substitution. The most common isoform, apo E3, has Cys112 and Arg158, whereas apo E2 has Cys158 and apo E4 has Arg112. These point mutations have a significant effect on the structural and physiological properties of apo E (Weisgraber, 1990; Wilson et al., 1994). The apo E genotype has been recognized as a susceptibility factor for AD (Corder et al., 1993; Schmechel et al., 1993; Strittmatter et al., 1993a,b). The frequency of the ⑀4 allele is significantly higher in sporadic and familial late-onset AD patients than in the general population: carriers of the ⑀4 allele have an ⬃15-fold higher risk of developing AD than noncarriers (Corder et al., 1993; Strittmatter et al., 1993a,b). These data suggest that the three apo E variants affect the rate of amyloidogenesis to a different extent. It has been shown that a direct interaction between apo E and A␤ peptide affects the in vitro formation and growth of amyloid plaques (Evans et al., 1995). We have recently determined, by molecular modeling, the existence of a hydrophobic fragment of sequence within the C-terminal domain of A␤ at residues 29 – 42, eliciting a tilted insertion with respect to a hydrophobic/ hydrophilic interface (Pillot et al., 1996). We first reported this type of peptide in viral glycoproteins, such as GP32 of simian immunodeficiency virus (SIV) (Horth et al., 1991). Those hydrophobic peptides can penetrate into a hydrophobic phase at an angle of 30 – 60° with respect to the plane of the interface, owing to the existence of a hydrophobicity gradient along their sequence (Brasseur, 1991; Brasseur et al., 1997). Through this oblique penetration, they destabilize the system in which they insert; for instance, viral tilted peptides destabilize the cell membrane and induce fusion (Horth et al., 1991). In agreement with sequence homology between the A␤ peptide and the viral fusion peptides (Currie et al., 1990), we showed that the A␤(29 – 42) tilted fragment can induce fusion of lipid vesicles and suggested that it could contribute to the A␤ neurotoxicity (Pillot et al., 1996). We further studied the interaction between this C-terminal fragment of A␤ and the three major apo E isoforms (Pillot et al., 1997). We showed that apo E2 and E3 can decrease liposome fusion and peptide aggregation induced by A␤(29 – 42) by direct interactions, whereas apo E4 has no effect. Similarly, apo A-I, whose structure is closely related to apo E, was able to reduce the fusogenic capacity of the SIV tilted peptide through a direct peptide–protein interaction (Srinivas et al., 1990; Martin et al., 1992; Pillot et al., 1997). Because apo A-I did not interact with the A␤ C-terminal domain and, conversely, apo E3 did not affect fusogenic properties of the SIV peptide (Pillot et al., 1997), we suggested that specific and complementary interactions might exist between some apolipoproteins and tilted peptides. However, the

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nature and molecular determinants of those interactions are unknown. In this article, we simulate the interactions between the amphipathic helices of apo E3 and apo A-I and the A␤ and SIV tilted peptides. We suggest that stable interactions occur when the hydrophobic bulky residues of the tilted peptides and the C-terminal helices of apo E3 and apo A-I are interacting. They could be responsible for protein selectivity and for decreasing fusogenic properties of tilted peptides. COMPUTATIONAL METHODS Molecular modeling of peptide conformation The method classically used to study peptide conformation is modified to take into account the dielectric constant variation and transfer of atoms from a hydrophobic to a hydrophilic environment characterizing a hydrophobic– hydrophilic interface (Brasseur, 1990; Brasseur et al., 1992). The total conformational energy is calculated as the sum of Van der Waals and electrostatic interactions, from the torsional potential energy and hydrophobic interactions quantified using an empirical equation for the free energy of solvation (Lins and Brasseur, 1995). Use of this corrective term in the energy field allows us to take water molecules into account implicitly and thus to obtain a better structure, as demonstrated for small molecules (Lins et al., 1996) and for the folding of small soluble proteins (Brasseur, 1995; Benhabiles et al., 1998). The lowest energy of the peptide is subsequently obtained by minimization using the Simplex method (Nelder and Mead, 1965), taking into account a hydrophobic– hydrophilic interface at each step of the calculation (Brasseur, 1990).

Molecular modeling of apolipoprotein helix–tilted peptide interaction This procedure is derived from that used to surround a drug with lipids (Brasseur et al., 1987). The initial position and orientation of both interacting fragments are defined such that helix axes are parallel. The position of the apolipoprotein helix stays still, while the interacting peptide (SIV or A␤) is allowed to translate along the x-axis and to rotate around itself and around the apolipoprotein helix. The sum of the Van der Waals, electrostatic, and hydrophobic energies between helical segments is then calculated and minimized. All calculations were performed on a Pentium processor, using PC-TAMMO⫹ (Theoretical Analysis of Molecular Membrane Organization) and PC-PROT⫹ (Protein Plus analysis) softwares. Graphs and molecular hydrophobicity potentials (MHP) (Brasseur, 1991) were drawn with the WinMGM program (Ab Initio Technology, France), and molecular lipophilic potential (MLP) surfaces (Fauche`re et al., 1988) were drawn using the CHIME program.

RESULTS We have reported that apo A-I and E3 decrease the liposome-destabilizing effect of the SIV tilted peptide and of the C-terminal domain of A␤ peptide, respectively (Pillot et al., 1996). Molecular interactions between those tilted peptides and the different amphipathic helices of apolipoproteins (Li et al., 1988; Brasseur et al., 1992; Jonas et al., 1993; Lins et al., 1995) are simulated here. Helices are the major structural determinants of apoliJ. Neurochem., Vol. 73, No. 2, 1999

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L. LINS ET AL. TABLE 1. Sequence of the peptides used in the present study

Protein fragment

Residue

Sequence

Apo E3 H1 Apo E3 H2 Apo E3 H3 Apo E3 H4 Apo E3 H5 Apo E3 H6 Apo E3 H7 Apo A-I A1 Apo A-I A2 Apo A-I A3 Apo A-I A4 Apo A-I A5 Apo A-I A6 Apo A-I A7 A␤ fragment SIV peptide

87–104 109–126 142–159 164–181 204–221 230–247 270–287 69–85 102–118 124–140 146–162 168–184 190–206 223–239 29–42

EETRARLSKELQAAQARL EDVCGRLVQYRGEVQAML RKLRKRLLRDADDLQKRL AGAREGAERGLSAIRERL QERAQAWGERLRARMEEM DEVKEQVAEVRAKLEEQA EDMQRQWAGLVEKVQAAV QEFWDNLEKETEGLRQE DDFQKKWQEEMELYRQK AELQEGARQKLHELQEK EEMRDRARAHVDALRTH DELRQRLAATLEALKEN AEYHAKATEHLSTLSEK ESFKVSFLSALEEYTKK GAIIGLMVGGVVIA GVFVLGFLGFLA

poprotein in lipids as well in solution, because the protein retains a large ratio of helicity in water (Jonas, 1992; Rosseneu et al., 1992). All the peptides used in this study are listed in Table 1. Based on the experimental data, we assumed a ratio of one A␤ tilted peptide to one helix of apo E3, as titration of the apo E–A␤ fragment complex suggests that one A␤ tilted peptide makes a stable association with one or two apo E3 ␣-helices (Pillot et al., 1997). For the sake of comparison, the same stoichiometry was used to calculate the interactions between apo A-I and SIV fusion peptide. Both tilted peptides can adopt ␣ and ␤ types of structures. In water, the structure is mainly ␤-sheet (Martin et al., 1991) or random coil (Coles et al., 1998). Increasing the solvent hydrophobicity, by adding sodium dodecyl sulfate (Coles et al., 1998), 2,2,2-trifluoroethanol (Pillot et al., 1996), or lipids (Martin et al., 1991), for example, increases the ␣-helical conformation. Fusogenic activity of A␤ and SIV tilted fragments increases with the helical content (Martin et al., 1991; Pillot et al., 1996), stressing the importance of that structure for the destabilizing capacity. Furthermore, it was recently shown by NMR that the fragment of the fusogenic domain of GP41 of human immunodeficiency virus corresponding to a tilted peptide (Brasseur, 1991), highly similar to the SIV tilted peptide in terms of hydrophobic/hydrophilic properties, is predominantly ␣-helical in a lipid-like environment (Chang et al., 1997). Finally, a helical conformation was assumed for both tilted peptides because calculations were performed imposing a ␤-strand and an ␣-helical conformation to the A␤(29 – 42) fragment and the two mutants that were shown to be nonfusogenic in vitro (Pillot et al., 1996). The orientation of the three peptides toward the interface do not correlate with variations of experimental results on liposome fusion when in ␤-strand, whereas they do in helical conformation (data not shown). There is no hydrophobicity gradient in the ␤-strand forms, and simulation of native residue 29 – 42 J. Neurochem., Vol. 73, No. 2, 1999

␤-strand peptide assembly with lipids does not indicate any perturbation of the lipid organization (data not shown), in contrast again with what happens with the helical form. In this ␣ conformation, the two mutants are, respectively, predicted to be oriented parallel and perpendicular to a hydrophobic– hydrophilic interface (Pillot et al., 1996). From those observations, we can assume that the ␣-helical conformation is the membrane-destabilizing structure of A␤ and SIV tilted fragments. The orientation of the A␤ C-terminal fragment and of the SIV fusion peptide at the interface is shown in Fig. 1. The most hydrophobic sides of helices are made of bulky residues (Met for the A␤ peptide and Phe for the SIV fusion peptide), which should disturb lipid organization. The interactions between apo E3 and the A␤ C-terminal domain and between apo A-I and the SIV peptide were calculated for both a parallel (C-N vs. C-N) and an anti-parallel (C-N vs. N-C) matching. Total and partial energies of interaction for each pair are listed in Tables 2 and 3. Simulations were triplicated starting from slightly different initial positions of interacting peptides. Variations around 10% in the relative energy values were observed (see Tables 2–5). For the apo E3–A␤ fragment interaction (Table 2), helix axes are mostly parallel, resulting in a maximal surface of interaction (Table 2 and Figs. 2A and 3A). The energy values reveal that the A␤ fragment interacts with apo E3 helices mainly through hydrophobic interactions. Those interactions often involve the bulky residue Met located on the destabilizing side of the tilted peptide (Fig. 2A). The short distance (⬃3.5– 4 Å) between the sulfur atom of the A␤ Met residue and the closest atom belonging to the apo E helix is representative of those interactions (Fig. 2A). Stabilizing interactions can also involve other hydrophobic residues of the A␤ fragment, as shown on Fig. 3A. The best interaction, in terms of hydrophobicity and interaction surface, is obtained with the last helix of apo E3, H7 (residues 270 –287; Fig. 2A). The assembly between the SIV peptide and the apo A-I helices is quite different (Table 3). On one hand, the parallelism of helix axes is less frequently observed, especially for the N-terminal part of apo A-I. This is highlighted by the distance between extremities of the interacting peptides (Fig. 3B) as compared with the apo E–A␤ system (Fig. 2A) and results in a decreased surface of interaction. This decrease is energetically balanced by strong polar interactions between polar residues in apo A-I helices and the N or C end of the SIV tilted peptide. When helices are parallel, they interact mainly through hydrophobic and electrostatic interactions. Both interactions can involve one or more of the three Phe residues of the destabilizing face of the SIV tilted peptide, with a mean distance between the Phe residue and the closest apo A-I residue of ⬃5– 6 Å (Fig. 2B). The best interaction, when helices are parallel, is obtained with the last helix of apo A-I, A7 (residues 223–239). This association is driven by hydrophobicity, and significant interac-

A␤–APOLIPOPROTEIN E INTERACTION

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FIG. 1. Simulation of the orientation of (A) the A␤(29 – 42) fragment and (B) the SIV fusion peptide at the interface (orange grid) between the hydrophobic (upper) and the hydrophilic (lower) phase. The N-terminal ends of the two tilted peptides are in the hydrophobic phase, with their C-terminal ends being in the hydrophilic phase below the interface.

tions exist between aromatic rings of the Phe residues of both helices (Fig. 2B). To test whether the protein selectivity of the apo A-I–SIV peptide and apo E–A␤ fragment interaction is due to a favorable energy of interaction and/or to residue specificity, we calculated cross-interactions, for instance, for apo E3–SIV peptide (Table 4) and apo A-I–A␤ tilted peptide (Table 5), that were experimentally shown to be ineffective (Pillot et al., 1997). It is obvious that most associations between helices of apo E3 and the SIV tilted peptide are not stabilized by hydrophobicity (Table 4), because only H3 (residues 142–159) and H4 (residues 164 –181) assemble with SIV fusion peptide through hydrophobic leucine–leucine interactions in only one orientation. Phe residues of the SIV peptide are never involved in those hydrophobic interactions (mean distance from Phe residue to closest apo E residue ⫽ 10 Å; Fig. 2C), and surfaces of interaction are drastically reduced because both axes of the interacting helices are tilted (mean distance between peptide N and C extremities ⫽ 15–20 Å; Fig. 2C).

Associations between apo A-I and the A␤ fragment are mostly hydrophobic (Table 5). Only one apo A-I segment, namely, residues 168 –184 (A5), is able to involve the Met residue of the A␤ tilted fragment in those interactions in both orientations and three other helices (A2, A3, and A6) in one orientation. No other helix involves this residue, as illustrated by a distance of 6 Å between the A␤ Met and the apo A-I Phe229 (Fig. 2D). DISCUSSION In this study, we analyzed by molecular modeling interactions between the amphipathic helices of apo E3 and the C-terminal domain of A␤ peptide, because we recently demonstrated that the membrane-destabilizing property of the A␤ fragment is significantly decreased by a direct interaction with apo E3 (Pillot et al., 1997). Those interactions were compared with the interaction between the SIV fusogenic tilted peptide and apo A-I, which reduces its fusogenic capacity (Martin et al., 1992; J. Neurochem., Vol. 73, No. 2, 1999

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L. LINS ET AL. TABLE 2. Interaction energies between apo E3 amphipathic segments and the A␤ fragment

Pairs H1/A␤ H1/A␤ H2/A␤ H2/A␤ H3/A␤ H3/A␤ H4/A␤ H4/A␤ H5/A␤ H5/A␤ H6/A␤ H6/A␤ H7/A␤ H7/A␤

par anti par anti par anti par anti par anti par anti par anti

Etot

EVdW

Epho

Ecb

Parallelism

A␤ Met35

⫺64 ⫾ 3 ⫺49 ⫾ 6 ⫺71 ⫾ 7 ⫺65 ⫾ 6 ⫺56 ⫾ 6 ⫺57 ⫾ 4 ⫺60 ⫾ 7 ⫺65 ⫾ 7 ⫺57 ⫾ 7 ⫺65 ⫾ 5 ⫺59 ⫾ 4 ⫺74 ⫾ 4 ⫺65 ⫾ 5 ⫺77 ⫾ 8

⫺12 ⫾ 2 ⫺19 ⫾ 3 ⫺23 ⫾ 2 ⫺18 ⫾ 2 ⫺15 ⫾ 3 ⫺8 ⫾ 2 ⫺11 ⫾ 3 ⫺12 ⫾ 1 ⫺16 ⫾ 2 ⫺12 ⫾ 3 ⫺17 ⫾ 1 ⫺18 ⫾ 1 ⫺13 ⫾ 3 ⫺10 ⫾ 2

⫺52 ⫾ 5 ⫺36 ⫾ 4 ⫺46 ⫾ 4 ⫺47 ⫾ 3 ⫺43 ⫾ 2 ⫺47 ⫾ 2 ⫺53 ⫾ 3 ⫺52 ⫾ 5 ⫺43 ⫾ 5 ⫺52 ⫾ 4 ⫺44 ⫾ 5 ⫺52 ⫾ 2 ⫺54 ⫾ 5 ⫺67 ⫾ 4

⫺0.5 ⫾ 1 6⫾2 ⫺2 ⫾ 2 0⫾2 2⫾2 ⫺2 ⫾ 0 4⫾1 ⫺1 ⫾ 1 2⫾1 ⫺1 ⫾ 2 2⫾0 ⫺4 ⫾ 2 2⫾2 0⫾2

⬇ 㛳㛳㛳㛳㛳㛳㛳㛳㛳㛳㛳㛳㛳

No Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes

par, parallel orientation of both segments (see text); anti, antiparallel orientation; Etot, total energy; EVdW, Van der Waals energy; Epho, hydrophobic energy; Ecb, electrostatic energy. Energies are mean ⫾ SE values, in kcal/mol, from three independent calculations. Parallelism indicates whether interacting peptides are parallel or not: 㛳, parallel; ⬇, tilt of ⬃20 – 40° between both axes; no, no parallelism with a tilt of ⬎40°. A␤ Met35 indicates whether or not the bulky residue(s) of the tilted peptide contributes to the interaction.

Pillot et al., 1997), and with the interactions between apo A-I and the A␤ fragment and between apo E3 and the SIV peptide, which are both unable to decrease fusogenic capacity in vitro (Pillot et al., 1997). The energy field used in the calculations is original in that it contains an empirical term simulating the hydrophobic effect (Lins and Brasseur, 1995) added to the classical forces. This additional term allows us to take water molecules into account implicitly. We recently demonstrated that using this energy field to calculate the conformation of small molecules yields better structures with respect to NMR data than classical energy fields (Lins et al., 1996). Comparison of energy values of the SIV–apo E3 interaction listed in Table 4 and fusogenic activity supports the proposal that a large contribution of electrostatic energy and a small surface of interaction between the tilted fragment and the apolipoprotein helices (poor Van der Waals energies) has no effect on fusogenic properties

of the tilted peptide (Fig. 2C). In contrast, the apo A-I– SIV (Table 3) and the A␤–apo E3 (Table 2) interactions are mostly hydrophobic and show good Van der Waals energies, so that our calculations can easily distinguish these interactions that are fusion-decreasing from the interaction between SIV and apo E3, at the level on their respective interaction energies. However, the interaction between apo A-I and A␤, which is a nonphysiological interaction, appears to be energetically similar to the apo A-I–SIV and apo E3–A␤ interactions. Apart from the energy analysis point of view, another important notion arising from our calculations is the steric complementarity between the apolipoprotein helix and the tilted peptide, together with the sides of the tilted fragment implicated in the interaction. A tilted destabilizing peptide is characterized by an asymmetric distribution of the hydrophobicity along the peptide when helical. The most hydrophobic side of the tilted peptides contains bulky hydrophobic residues (A␤:

TABLE 3. Interaction energies between apo A-I amphipathic segments and the SIV fusion peptide Pairs A1/SIV A1/SIV A2/SIV A2/SIV A3/SIV A3/SIV A4/SIV A4/SIV A5/SIV A5/SIV A6/SIV A6/SIV A7/SIV A7/SIV


Etot

EVdW

Epho

Ecb

Parallelism

SIV Phe

⫺93 ⫾ 5 ⫺138 ⫾ 10 ⫺66 ⫾ 5 ⫺83 ⫾ 7 ⫺71 ⫾ 5 ⫺70 ⫾ 5 ⫺74 ⫾ 6 ⫺65 ⫾ 6 ⫺64 ⫾ 7 ⫺56 ⫾ 5 ⫺65 ⫾ 7 ⫺87 ⫾ 5 ⫺79 ⫾ 6 ⫺97 ⫾ 7

⫺18 ⫾ 3 ⫺6 ⫾ 2 ⫺12 ⫾ 2 ⫺9 ⫾ 2 ⫺3 ⫾ 1 ⫺9 ⫾ 1 ⫺15 ⫾ 2 ⫺6 ⫾ 2 ⫺15 ⫾ 2 ⫺19 ⫾ 2 ⫺6 ⫾ 1 ⫺4 ⫾ 2 ⫺12 ⫾ 2 ⫺13 ⫾ 2

⫺50 ⫾ 6 ⫺13 ⫾ 2 ⫺50 ⫾ 4 ⫺64 ⫾ 5 ⫺6 ⫾ 1 ⫺24 ⫾ 2 ⫺16 ⫾ 2 ⫺3 ⫾ 1 ⫺50 ⫾ 4 ⫺37 ⫾ 5 ⫺11 ⫾ 1 ⫺8 ⫾ 1 ⫺70 ⫾ 8 ⫺73 ⫾ 5

⫺25 ⫾ 3 ⫺119 ⫾ 13 ⫺4 ⫾ 1 ⫺10 ⫾ 3 ⫺62 ⫾ 6 ⫺37 ⫾ 2 ⫺44 ⫾ 4 ⫺56 ⫾ 4 1⫾1 0⫾2 ⫺48 ⫾ 6 ⫺75 ⫾ 5 3⫾1 ⫺11 ⫾ 2

⬇ No ⬇ ⬇ ⬇ ⬇ 㛳 No 㛳㛳㛳㛳㛳㛳

Yes No Yes Yes No No No No No Yes Yes Yes Yes Yes

See Table 2 for definitions. SIV Phe indicates whether or not the bulky residue(s) of the tilted peptide contributes to the interaction.

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FIG. 2. Association between (A) A␤ fragment and the residue 270 –287 apo E3 segment, (B) SIV fusion peptide and the residue 223–239 apo A-I segment, (C) SIV fusion peptide and the residue 142–159 apo E3 segment, and (D) A␤ fragment and the residue 223–239 apo A-I segment. Distances (see text) are expressed in angstroms.

1 Met; SIV: 3 Phe) that should have a steric destabilizing effect when inserted in a hydrophobic– hydrophilic interface. Thus, an interaction occurring between this face of the tilted peptides and the hydrophobic face of the apolipoprotein amphipathic segments should be largely more efficient in decreasing the fusogenic activity than an interaction implicating the other side. By comparing the interaction of apo A-I and SIV (Table 3) and apo A-I and A␤ (Table 5), and especially the interaction of the last helix of apo A-I (A7), we observe that hydrophobic interactions with good Van der Waals energies occur in both cases but that the destabilizing face of SIV, i.e., that containing the Phe residues, is implicated in the interaction with the last helix of apo A-I, whereas the Met residue, i.e., the “fusion-active” face, of A␤ is not. Figure 2B shows a strong complementarity between the last helix of apo A-I, at residues

223–239, and Phe residues of the SIV peptide by cycle– cycle interactions inducing favorable interactions between ⌸ orbitals. The C-terminal part of apo E3 interactions also displays steric complementarity and hydrophobic interactions involving the Met residue in their assembly with the A␤ fragment (Fig. 2A). In both cases, those interactions are not observed in the ineffective complexes. For instance, the apo E3 C-terminal helix does not contain aromatic residues such as Phe, which generate cycle– cycle interactions, energetically and geometrically favorable in the association between the last helix apo A-I and the SIV peptide. The A␤ fragment makes hydrophobic interactions with apo A-I helix 7, but the Met residue of the A␤ fragment does not participate (Fig. 2D). The involvement of the C-terminal domain and notably of the last helix of apo E3 is further supported by the J. Neurochem., Vol. 73, No. 2, 1999

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FIG. 3. Association between the A␤ fragment and the residue 87–104 apo E3 segment (A) and between the SIV fusion peptide and the residue 146 –162 (B) and residue 168 –184 (C) apo A-I segments. Distances (see text) are expressed in angstroms.

experimental fact that only the C-terminal domain of apo E3 decreases the extent of vesicular fusion induced by the A␤(29 – 42) fragment, whereas the residue 129 –169 N-terminal domain has no effect (Table 6). The decrease is due to direct and specific interactions between both fragments, as shown by gel electrophoresis (Pillot et al., 1999): After a 1-h incubation at 25°C of the apo E3 C-terminal fragment with the A␤(29 – 42) peptide, a band with an apparent mass of 17–18 kDa corresponding to stable complexes containing one apo E fragment and four to five amyloid peptide chains is obtained. No complex was formed with the SIV fusion peptide, stressing the specificity of the apo E3–A␤ interaction. Conversely, we previously demonstrated that the A␤ tilted fragment decreases the lipid-binding affinity of apo E3 (Pillot et al., 1997). This lipid association capacity was attributed to the C-terminal domain of apo E (De Pauw et al., 1997). The Trp residue of apo E3 helix 5 (residues 204 –221) and helix 7 (residues 270 –287) (Fig. 2A) is involved in hydrophobic interactions with the A␤ fragment, in agreement with fluorescence data that indi-

cate a more hydrophobic environment of apo E3 Trp residues when associated with the A␤ tilted peptide (Pillot et al., 1997). Taking all those observations into account, we propose that the interactions between the A␤ tilted peptide and apo E3 helices and, by extension, between the apo A-I and SIV tilted peptide involve essentially the C-terminal domain and, very probably, the last helix of the apolipoproteins. Previous reports, using synthetic peptides or deletion mutants (Sparrow et al., 1992; Holvoet et al., 1995), had shown that the last helical segment of apo A-I and apo E3 is responsible for the initial binding of the protein to lipids. As the last helix of those apolipoproteins is the most hydrophobic segment (Brasseur et al., 1992), we can assume that it should trigger any interaction with hydrophobic molecules such as lipids or tilted fragments. When initiated, interactions between the tilted peptide and other apolipoprotein helices might occur. For apo A-I only the C-terminal helices present a favorable hydrophobic energy when interacting with SIV. Taking into account the similar energies of interaction for all the

TABLE 4. Interaction energies between apo E3 amphipathic segments and the SIV peptide Pairs H1/SIV H1/SIV H2/SIV H2/SIV H3/SIV H3/SIV H4/SIV H4/SIV H5/SIV H5/SIV H6/SIV H6/SIV H7/SIV H7/SIV


Etot

EVdW

Epho

Ecb

Parallelism

SIV Phe

⫺68 ⫾ 5 ⫺89 ⫾ 7 ⫺71 ⫾ 6 ⫺105 ⫾ 7 ⫺143 ⫾ 17 ⫺67 ⫾ 10 ⫺113 ⫾ 10 ⫺71 ⫾ 5 ⫺116 ⫾ 9 ⫺161 ⫾ 18 ⫺164 ⫾ 15 ⫺137 ⫾ 9 ⫺119 ⫾ 10 ⫺144 ⫾ 11

6⫾1 ⫺12 ⫾ 2 ⫺2 ⫾ 1 ⫺1 ⫾ 2 6⫾2 ⫺13 ⫾ 3 5⫾1 ⫺17 ⫾ 2 ⫺3 ⫾ 0 16 ⫾ 3 ⫺7 ⫾ 1 ⫺10 ⫾ 1 10 ⫾ 1 0⫾1

⫺30 ⫾ 2 ⫺25 ⫾ 3 ⫺23 ⫾ 4 ⫺6 ⫾ 1 ⫺24 ⫾ 3 ⫺47 ⫾ 7 2⫾1 ⫺51 ⫾ 3 ⫺5 ⫾ 1 ⫺10 ⫾ 1 ⫺6 ⫾ 2 ⫺15 ⫾ 2 3⫾2 ⫺12 ⫾ 3

⫺44 ⫾ 3 ⫺52 ⫾ 4 ⫺46 ⫾ 3 ⫺98 ⫾ 8 ⫺125 ⫾ 13 ⫺7 ⫾ 2 ⫺120 ⫾ 13 ⫺3 ⫾ 1 ⫺108 ⫾ 9 ⫺167 ⫾ 20 ⫺151 ⫾ 14 ⫺112 ⫾ 10 ⫺132 ⫾ 14 ⫺132 ⫾ 12

⬇ ⬇ ⬇ No No ⬇ ⬇ ⬇ ⬇ ⬇ No ⬇ No No

No No No No No No No No No No No No No No

See Tables 2 and 3 for definitions.



765

TABLE 5. Interaction energies between apo A-I amphipathic segments and the A␤ fragment Pairs A1/A␤ A1/A␤ A2/A␤ A2/A␤ A3/A␤ A3/A␤ A4/A␤ A4/A␤ A5/A␤ A5/A␤ A6/A␤ A6/A␤ A7/A␤ A7/A␤


Etot

EVdW

Epho

Ecb

Parallelism

A␤ Met35

⫺70 ⫾ 5 ⫺79 ⫾ 6 ⫺80 ⫾ 9 ⫺65 ⫾ 4 ⫺48 ⫾ 5 ⫺61 ⫾ 6 ⫺51 ⫾ 4 ⫺42 ⫾ 4 ⫺77 ⫾ 8 ⫺66 ⫾ 5 ⫺53 ⫾ 5 ⫺45 ⫾ 6 ⫺75 ⫾ 6 ⫺95 ⫾ 8

⫺12 ⫾ 3 ⫺15 ⫾ 1 ⫺15 ⫾ 2 ⫺4 ⫾ 1 ⫺14 ⫾ 2 ⫺15 ⫾ 2 ⫺14 ⫾ 1 ⫺11 ⫾ 1 ⫺21 ⫾ 3 ⫺14 ⫾ 2 ⫺18 ⫾ 3 ⫺10 ⫾ 2 ⫺11 ⫾ 1 ⫺14 ⫾ 2

⫺54 ⫾ 3 ⫺62 ⫾ 7 ⫺66 ⫾ 8 ⫺65 ⫾ 4 ⫺36 ⫾ 3 ⫺44 ⫾ 3 ⫺36 ⫾ 4 ⫺32 ⫾ 5 ⫺53 ⫾ 5 ⫺51 ⫾ 4 ⫺39 ⫾ 2 ⫺34 ⫾ 4 ⫺66 ⫾ 5 ⫺80 ⫾ 7

⫺4 ⫾ 1 ⫺2 ⫾ 2 1⫾1 4⫾1 2⫾0 ⫺2 ⫾ 1 1⫾1 1⫾0 ⫺3 ⫾ 2 ⫺1 ⫾ 1 4⫾2 1⫾1 2⫾1 ⫺1 ⫾ 2

㛳㛳 ⬇ 㛳㛳 No 㛳㛳㛳㛳 ⬇ 㛳㛳㛳

No No Yes No Yes No No No Yes Yes No Yes No No

See Table 2 for definitions.

apo E3 segments (Table 2), we suggest that after the initiation of the interaction by H7, other helices could interact with A␤. Those helices should be C-terminal helices (H4 –H6), whose physiological role is to bind lipids, i.e., hydrophobic molecules, but also the N-terminal helices (H1–H3), and this only when H7 is present. Those considerations suggest that interactions of apolipoproteins with hydrophobic tilted peptides could be similar to those in apolipoprotein–lipid complexes, such as the complexes found in interstitial fluid in vivo (Sloop et al., 1987). They consist of one molecule of apolipoprotein and ⬃10 molecules of lipids. In the apolipoprotein– tilted peptide complexes, bulky residues of tilted peptides should be involved in hydrophobic interactions with the apolipoprotein helices; otherwise, lateral steric hindrance could destabilize the multimeric complexes. Conversely, when the most hydrophobic face of the tilted helix is involved in the interaction with apolipoprotein helical segment, its other face, containing smaller residues such as Gly, Ala, or Leu, could create self-associations of tilted peptides. Calculations support the proposal that such complexes would be energetically stable, as shown in Fig. 4A, left, for the A␤ fragment and apo E3. Hydrophobic interactions are the major force holding the segments together. Calculation of the MHP of this TABLE 6. Effects of apo E3 domains on lipid-mixing properties of A␤(29 – 42)

Apo E3(1–299) Apo E3 (200–299) Apo E (129–169)

Apo E/A␤(29–42) ratio (mol/mol)

Lipid mixing (%)

0 1/100 0 1/100 0 1/100

55a 35a 40 22 42 40

Lipid mixing induced by the amyloid fragment (14 ␮M) was monitored as previously described (aPillot et al., 1996) and was measured after 10 min of incubation.

complex, giving the hydrophobic and hydrophilic environment around the molecule, shows that the outer surface is mostly hydrophilic (green surface on Fig. 4A, right), whereas the core is hydrophobic (orange surface in Fig. 4A, right). This configuration is observed for any soluble protein. For example, the MHP of the globular N-terminal domain of apo E3, whose x-ray structure is known (Wilson et al., 1991), is represented in Fig. 4B, and this is clearly different from what is observed for transmembrane proteins, such as bacteriorhodopsin (Henderson et al., 1990) (Fig. 4C). In the latter, the hydrophobicity potential shows that the outer envelope is essentially hydrophobic, with a few hydrophilic pockets at the top of the protein for the domains in contact with the polar headgroups of the lipids (Fig. 4C, right). This suggests that apo E3 could solubilize the Cterminal domain of A␤ peptide, which is critical for amyloid fibril formation (Lansbury et al., 1995) and for lipid destabilization (Pillot et al., 1996). The solubilization effect is further supported by the significant decrease of the accessible hydrophobic surface when the A␤ Cterminal fragment is associated with the last helix of apo E3, as shown by calculation of the MLP for the two isolated fragments (Fig. 5A and B) as compared with the pair (Fig. 5C). Apo E consists of two domains: the N-terminal region, which is responsible for the binding of apo E to the low-density lipoprotein receptor (Aggerbeck et al., 1988; Wilson et al., 1991; De Pauw et al., 1995), and the C-terminal region, which mediates the binding of apo E to the surface of lipoproteins (Weisgraber, 1990; Westerlund and Weisgraber, 1993; De Pauw et al., 1995). Three apo E isoforms can occur in vivo, namely, apo E2, E3 (the most common allele), and E4. Apo E2 and E4 differ from apo E3 by point mutations in the N-terminal domain: an R158C mutation in apo E2 and a C112R mutation in apo E4. The C-terminal region is identical in the three isoforms. This is in apparent contradiction with the above discussion because our calculations suggest that the interaction between the A␤ fragment and apo E3 J. Neurochem., Vol. 73, No. 2, 1999

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FIG. 4. A: Front view of computed complex formed between the four C-terminal apo E3 helices (H4 –H7 in Table 1) and four A␤(29 – 42) fragments (left). Helices are red cylinders. MHP calculation (Brasseur, 1991) is carried out on the complex in the same orientation (right). Hydrophobic areas are orange, and hydrophilic ones are green. B: Front view of the apo E3 N-terminal x-ray structure (left) and its MHP (right). Same representation as in A. C: Front view of the bacteriorhodopsin x-ray structure (left) and its MHP (right). Same representation as in A.

involves its C-terminal domain, although the three apo E isoforms do not display the same inhibitory effect on the fusogenic properties of the A␤ fragment (Pillot et al., 1997). Apo E2 and E3 decreased fusion through direct interaction, whereas apo E4 had no effect, and no protein–peptide interaction was observed (Pillot et al., 1997). However, it has been demonstrated by x-ray crystallography that point mutations in the apo E N-terminal J. Neurochem., Vol. 73, No. 2, 1999

domain can significantly affect the three-dimensional organization of the C-terminal domain of the protein (Wilson et al., 1994; Dong et al., 1996). Lipid-binding properties also differ between isoforms, because the residue at position 112 influences the distribution of isoforms among the various lipoprotein classes (Weisgraber, 1990). These effects suggest a significant cooperativity between N- and C-terminal domains of apo E. The


767

FIG. 5. MLP calculations on the molecular surface of (A) the A␤ C-terminal fragment, (B) the residue 270 –287 apo E3 segment, and (C) their association. Color scale is as follows: hydrophobic, orange; neutral, white; hydrophilic, green. MLP values are calculated according to the equation of Fauche`re et al. (1988).

experimentally observed difference between those isoforms should thus be explained by a different conformation inducing a different behavior of the C-terminal domain in the three isoforms due to the N and C domain cooperativity. In conclusion, our calculations suggest that hydrophobic interactions could occur between the apolipoprotein C-terminal domain and tilted hydrophobic helices and could have an inhibitory effect on the fusogenic process of the latter, in agreement with experimental data. This process is apolipoprotein-specific, owing to the complementarity between the bulky hydrophobic residues in tilted peptides and the hydrophobic residues in apolipoprotein amphipathic segments. Finally, if the A␤ C-terminal domain is involved in neurotoxicity, these results could open the way to search for therapeutic agents (peptides or drugs) that would complement A␤ hydrophobic residues crucial for the destabilizing effect of this domain. This holds true for the SIV fusogenic peptide. It could be an easier target, because the blood– brain barrier should not be crossed. Acknowledgment: R.B. is Research Director at the National Funds for Scientific Research of Belgium. This work was supported by the “Interuniversity Poles of Attraction Programme–Belgian State, Prime Minister’s Office—Federal Office for Scientific, Technical and Cultural Affairs” contract P.4/03, the Loterie Nationale, and the National Fund for Scientific Research of Belgium and the Fonds de la Recherche Scientifique Me´dicale, program 3.4571.98.

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