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Neurotoxic assemblies of the amyloid β-protein (Aβ) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Aβ assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Aβ(1–40) and Aβ(1–42). The results revealed a 10-residue, protease-resistant segment, Ala21–Ala30, in both peptides. Remarkably, the homologous decapeptide, Aβ(21–30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24–Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a β-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24–Lys28 region of Aβ nucleates the intramolecular folding of Aβ monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Aβ(1–40) and Aβ(1–42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Aβ(1–42) production. Our results suggest that therapeutic approaches targeting the Val24–Lys28 turn or the Aβ(1–42)-specific C-terminal fold may hold promise.
The amyloid β-protein (Aβ) is a normal, soluble component of human plasma and cerebrospinal fluid (Haass et al. 1992; Seubert et al. 1992; Shoji et al. 1992). Aβ exists predominantly as a 40- or 42-residue peptide (for review, see Teplow 1998). Historically, the assembly of Aβ into amyloid fibrils was thought to initiate a pathogenic cascade resulting in AD (the amyloid cascade hypothesis) (Hardy and Allsop 1991). However, progressively smaller assemblies have been discovered (Oda et al. 1995; Harper et al. 1997; Walsh et al. 1997; Lambert et al. 1998; Bitan et al. 2003a), and each has been found to be neurotoxic (Lambert et al. 1998; Hartley et al. 1999; Walsh et al. 1999; Sun et al. 2003; Taylor et al. 2003). This accumulating evidence supports a revision of the amyloid cascade hypothesis such that Aβ assembly into neurotoxic oligomers, and not into fibrils, is the seminal event in AD pathogenesis (Haass and Steiner 2001; Klein et al. 2001, 2004; Kirkitadze et al. 2002; Walsh et al. 2002). If the hypothesis is true, then preventing the folding of nascent Aβ monomer into toxic conformers or oligomers would have therapeutic benefit.
Certain regions of Aβ exert strong control over assembly kinetics and biological activity. The dipeptide Ile41–Ala42 at the C terminus of Aβ, which distinguishes Aβ(1–42) from Aβ(1–40), is responsible for distinct biophysical (Jarrett et al. 1993; Bitan et al. 2003a), physiologic (Klein et al. 2004), and clinical (Iwatsubo et al. 1995) behaviors of the longer peptide. These behaviors include oligomerization into pentamer/hexamer units (paranuclei) (Bitan et al. 2003a), nucleation of amyloid formation from shorter variants such as Aβ(1–39) and Aβ(1–40) (Jarrett et al. 1993), formation of Aβ-derived diffusible ligands (ADDLs) (Oda et al. 1995; Lambert et al. 1998), early deposition in senile plaques in Down's syndrome patients (Iwatsubo et al. 1995), and strong linkage to AD (Younkin 1995). Amino acid substitutions within and adjacent to the central hydrophobic cluster (CHC) of Aβ, Leu17–Ala21, cause cerebral amyloid angiopathy and AD-like diseases (Levy et al. 1990; Kamino et al. 1992; Hendriks and Vanbroeckhoven 1996; Tagliavini et al. 1999; Grabowski et al. 2001; Nilsberth et al. 2001). In vitro studies have probed the biophysical consequences of these mutations. For example, the Glu22Gln substitution increases both the rates of nucleation and elongation of Aβ fibrils (Wisniewski et al. 1991; Teplow et al. 1997) and the Glu22Gly substitution facilitates protofibril formation (Nilsberth et al. 2001; Päiviö et al. 2004). Met35 may be important both in Aβ-associated redox chemistry (Butterfield 2002) and in the peptide assembly (Snyder et al. 1994; Seilheimer et al. 1997; Watson et al. 1998; Butterfield 2002; Hou et al. 2002a; Palmblad et al. 2002). Recent studies suggest that formation of methionine sulfoxide (Met[O]) or methionine sulfone (Met[O2]) block oligomerization of Aβ(1–42), preventing fibril assembly (Hou et al. 2002b; Palmblad et al. 2002; Bitan et al. 2003b). Taken together, these data have revealed how small changes in Aβ primary structure can alter intermolecular interactions among Aβ monomers.
To examine assembly-dependent features of intramolecular peptide organization, Aβ secondary structure characteristics have been determined spectroscopically. The conformational changes occurring during fibril assembly involve random coil (RC)α-helix, RCβ-strand, and α-helixβ-strand transitions (Barrow et al. 1992; Soto et al. 1995; Sticht et al. 1995; Coles et al. 1998; Shao et al. 1999; Walsh et al. 1999; Zagorski et al. 2000; Zhang et al. 2000; Kirkitadze et al. 2001). The involvement of turn elements early in fibril formation is less clear. Studies of Aβ fragments, including Aβ(19–28) (Gorevic et al. 1987; Sorimachi et al. 1990), Aβ(15–28) (Gorevic et al. 1987), and Aβ(25–35) (Laczko et al. 1994), suggest turns are present. However, no turns were found in studies of Aβ(10–35) fibrils (Benzinger et al. 2000). In contrast, modeling based on established examples of β-helical conformation, and on hydrophobic and hydrogen bonding effects on protein folding, suggests a β-turn at Val24–Asn27 (Lazo and Downing 1998). In silico modeling of fibrils from Aβ(1–43) reveals a β-turn at Gly25–Lys28 (George and Howlett 1999). Solid-state NMR studies of Aβ(1–40) fibrils suggest a bend at Gly25–Gly29 (Petkova et al. 2002).
As illustrated above, a deepening understanding of Aβ peptide oligomerization and fibril formation is being obtained. The aim of the work reported here was to establish how these important assembly processes are initiated at the monomer level. To do so, we probed the conformation of Aβ monomer using limited proteolysis (Fontana et al. 1997; Hubbard 1998) in combination with mass spectrometry. This approach is useful in the study of conformational changes in proteins that have a strong propensity to aggregate (Kheterpal et al. 2001; Polverino de Laureto et al. 2003; Monti et al. 2004). Results suggest, in both Aβ(1–40) and Aβ(1–42), that a nucleus for intramolecular folding exists within the decapeptide region Ala21–Ala30. Solution-state NMR studies of the corresponding Aβ(21–30) decapeptide revealed a turn element formed by residues Val24–Lys28 and provided a resolved structure of the nucleus.
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The data presented here are consistent with the postulation that one of the initial events in pathologic Aβ monomer folding is an intramolecular nucleation event in which the Val24–Lys28 region forms an unusual turn conformation centered at Gly25–Asn27 (Fig. 6). It is possible that a Val24–Lys28 five-residue turn may result from (i,i+1) double turns (Hutchinson and Thornton 1994), which consist of five residues such that residues 1–4 (Val24– Gly25–Ser26–Asn27) and residues 2–5 (Gly25–Ser26– Asn27–Lys28) form turns. The central residues in the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys turns (Gly-Ser and Ser-Asn, respectively) have high overall positional potential to form the central residues of β-turns (Hutchinson and Thornton 1994). Multiple elements thus may contribute to the formation and stabilization of the turn in Aβ monomer, including: (1) the intrinsic propensity of Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form β-turns (Hutchinson and Thornton 1994); (2) hydrophobic interactions between Val24 and Lys28 side chains; and (3) long-range Coulombic interaction between Lys28 and either Glu22 or Asp23. The existence of two conformational families may reflect competition between the carboxylate anions of these two latter amino acids for the NH3 + group of Lys28.
The observations of a folding nucleus within the Ala21– Ala30 region of full-length Aβ and a homologous structure in the isolated Aβ(21–30) decapeptide are consistent with previous work showing that peptide fragments encompassing the folding nuclei of globular proteins, by themselves, are structured (Neira and Fersht 1996). Furthermore, the structures found in the folding nuclei are similar to those found in the holoproteins (Neira and Fersht 1996). Our data are consistent with the Aβ folding nucleus appearing in the monomer form of the peptide. The data reported here, and by others (Kheterpal et al. 2003), demonstrates that nonmonomer forms of Aβ, including oligomers, protofibrils, and fibrils, show backbone peptide-bond protection. Aβ amyloid formation, and amyloid formation by other proteins, is thought to be driven to a significant degree by hydrophobic interactions among peptide segments that are solvent-exposed in the partially unfolded state (Dobson 1999). We observed that hydrophobic regions in Aβ(1–40), including the CHC and the C terminus, were accessible to proteolysis (Fig. 3) and therefore not involved either in intra- or intermolecular folding interactions. Previously, we demonstrated that both LMW Aβ(1–40) and Aβ(1–42) exist in equilibria among monomers and low-order (low n) oligomers: n=2–4 for Aβ(1–40) and n=5,6 for Aβ(1–42) (Bitan et al. 2003a). These equilibria are extremely rapid, establishing themselves from the pure monomer state with time constants <<1 min (Bitan et al. 2003a). We also showed that some structural order exists in chemically-stabilized oligomers (Bitan et al. 2003a). Hydrogen exchange experiments have confirmed that protection factors in oligomers differ significantly from monomers (Kheterpal et al. 2003). A consideration of these published data, and the results reported here, suggests that a simple and logical explanation for our data is that proteolysis revealed peptide bond exposure primarily in the monomer state and that this state, following Le Châtelier's principle (Le Châtelier 1884), was rapidly repopulated by disassembly of low-order oligomers during the digestion procedure.
We note that theVal39–Ala42 region in LMWAβ(1–42) is protease-resistant in the absence of denaturant (Fig. 3). This resistance in the monomer state requires structure, which may involve Gly–Gly turns (Grant et al. 2000; Gibbs et al. 2002). β-Helix models of fibrils formed by full-length Aβ (Lazo and Downing 1998) and Aβ(34–42) (Lazo and Downing 1999) contain Gly37–Gly38 turns. Importantly, in silico studies of Aβ monomer folding have shown that a Gly37–Gly38 turn forms in Aβ(1–42) but not in Aβ(1–40) (Urbanc et al. 2004).
Our work offers a mechanistic explanation for the effects of disease-associated amino acid substitutions clustered in the Ala21–Ala30 region of Aβ. Five such substitutions have been reported, all of which are found between Ala21 and Asp23. The mutations giving rise to the substitutions are named after the ethnicities of the kindreds in which they were identified and include the Flemish (Ala21Gly) (Cras et al. 1998), Arctic (Glu22Gly) (Kamino et al. 1992; Nilsberth et al. 2001), Dutch (Glu22Gln) (Levy et al. 1990), Italian (Glu22Lys) (Bugiani et al. 1998), and Iowa (Asp23Asn) (Grabowski et al. 2001) mutations. The existence of long-range stabilizing Coulombic interactions between Glu22/Asp23 and Lys28, and the involvement of the aminobutyl side chain of Lys28 in hydrophobic interactions, offer an explanation for how these AD- and cerebral amyloid angiopathy-linked amino acid substitutions cause pathology—they alter the structure or stability of the folding nucleus. How these alterations affect the biophysical and biological properties of the peptide segment defining the folding nucleus is difficult to determine pre facto. As with any mutation in a protein structural gene, we would predict a priori that substitutions could enhance, inhibit, or have no effect on peptide segment-specific properties. In practice, substitutions in the Aβ(21– 23) region have been shown to cause aberrant AβPP processing (Aβ anabolism) (Hardy 1997), inhibition of Aβ degradation and clearance (Aβ catabolism) (Tsubuki et al. 2003), increased peptide neurotoxicity (Melchor et al. 2000), and changes in Aβ assembly kinetics or pathway. For example, the Arctic mutation leads to enhanced Aβ protofibril formation but decreased overall Aβ production (Nilsberth et al. 2001). All three Glu22 substitutions lead to enhanced fibril assembly on the surface of human cerebrovascular smooth muscle cells, where amyloid is deposited in cerebral amyloid angiopathy (Melchor et al. 2000).
Conceptually, the biophysical basis of mutation-induced, accelerated Aβ self-association has been thought to be the enhanced ability of the altered amino acid side chain to incorporate into pathologic structures. The data presented here provide deeper mechanistic insights into the assembly process and suggest alternative explanations to explain mutant phenotypes. Specifically, primary structure changes within the Ala21–Ala30 decapeptide segment alter pathways of nucleation. These alterations can either be quantitative (kinetic) or qualitative (altering pathway choice). Substitutions at Glu22 or Asp23 would affect long-range interactions of these amino acids with Lys28, changing the propensity of the Aβ monomer to populate conformational family I or II. The Italian, Dutch, and Arctic mutations, for example, would destabilize the Coulombic Glu22:Lys28 interaction, shifting the folding pathway away from family I toward family II. If fibril formation from family II conformers were more favorable energetically than from family I conformers, disruption of Glu22:Lys28 interactions within the folding nucleus could produce the counterintuitive result of increased fibril formation. Results from recent discrete molecular dynamics simulations of Aβ(21–30) folding support this postulation (Borreguero et al. 2005). These simulations show that electrostatic interactions between Asp23 and Lys28 are favored over those involving Glu22 and Lys28 in conformers with a propensity for fibril formation. In fact, experimental studies have shown that an engineered lactam bridge between Asp23 and Lys28 increases the Aβ(1–40) fibrillogenesis rate by ≈ 1000-fold (Sciarretta et al. 2005). In addition to those substitutions enhancing nucleation, certain substitutions might inhibit monomer nucleation. These latter mutations would be nonpathogenic, and therefore it is unlikely they would be identified clinically using strategies designed to recognize pathologic phenotypes.
Wurth et al. (2002) have used an unbiased genetic approach to identify amino acid substitutions in Aβ(1–42) that reduced the aggregation of Aβ(1–42)-green fluorescent protein (GFP) fusion proteins in Escherichia coli. This approach revealed six strains in which substitutions in the Aβ(21–30) region occurred: GM71, Glu22Gln, Ala30Pro; GM57, Ser26Phe; GM13, Val24Gly; GM11 and GM16, Val24Ala; and GM9, Ser26Pro. Unfortunately, all of these strains expressed fusion proteins in which amino acid substitutions also occurred outside the Aβ(21–30) region, complicating structure–activity correlation. It is provocative, however, that the mutations that were observed involved residues predicted by our structural model to have significant influence on Aβ folding, including Glu22 (Coulombic interactions), Val24 (hydrophobic interactions), and Ser26 (turn formation). Williams et al. (2004) used a different approach, scanning proline mutagenesis, to study secondary structure within fibrils formed by Aβ(1–40) and to derive a model of the Aβ(1–40) protofilament. This model differs from those derived by solid-state NMR (Petkova et al. 2002), especially with respect to the location of turns. Rather than predict a turn in the 25–29 region, Williams et al. predict two turns, one centered at Glu22– Asp23 and the other centered at Gly29–Ala30. Additional studies will be required to reconcile these differing models of fibril structure. Our data are relevant to the earliest folding events in Aβ self-assembly and suggest that relatively minor rearrangement is necessary to produce fibrils arranged as suggested by Petkova et al. (2002). However, our model does not exclude the possibility of more substantial rearrangement, as would be required to satisfy the model of Williams et al. (2004).
We have focused here on full-length Aβ proteins and the Aβ(21–30) peptide. Zhang et al. (2000) examined the structure of an Aβ fragment intermediate in size, Aβ(10–35). This peptide has been an object of study because it has been found to be “plaque competent,” i.e., it provides a source of monomers for elongation of pre-existent fibrils (Lee et al. 1995). Based on distance constraints from NOESY data, Zhang et al. derived two equally probable families of structures for Aβ(10–35). The key feature of these families was a collapsed coil structure formed by the CHC, accompanied by “a series of loops and turns condensed about the CHC foundation.” To assess any similarities or differences among the structures derived by Zhang et al. and those presented here, we superimposed the backbones of our families I and II on their respective regions within Aβ(10–35) (data not shown). We found that the superimposition of the backbone of family I from Asp23 to Lys28 with that of Aβ(10–35) (PDB ID 1HZ3) produced excellent agreement, particularly along the SN sequence.
We are aware of one solution-state NMR study examining full-length, unmodified Aβ in cosolvent-free, aqueous solution (Hou et al. 2004). Using Aβ(1–40) and Aβ(1–42) in phosphate-buffered solution (pH 7.2), Hou et al. (2004) concluded that both proteins contain turnor bend-like structures at Asp7–Glu11 and Phe20–Ser26. The only two regions of full-length Aβ that we found contained four or more contiguous protease-resistant peptide bonds were Asp7–Glu11 and Ala21–Ala30. The former region matches precisely that defined by Hou et al. The region Phe20–Ser26 differs somewhat from, but is largely contained within, the Ala21–Ala30 segment we defined. The lack of perfect correspondence between the two data sets may be related to differing experimental conditions. To delay β-sheet formation, Hou et al. performed their experiments at 5°C. At this temperature, medium-range NOEs throughout the Phe20–Ala30 region were observed and it was speculated that the region exists as an ensemble of rapidly interconverting random structures with kink- or bend-like propensities (Hou et al. 2004). At 25°C, the temperature used in our limited proteolysis studies, it is possible that a turn region similar to family I or II might be populated more frequently.
One prediction of the monomer nucleation model we have proposed is that the nucleation center should be present in fibrils, the end-stage assemblies of Aβ self-association. Although a complete three-dimensional structure of Aβ fibrils has not yet been determined (Tycko 2004), evidence from a number of groups supports the existence of a turn or bend structure. Early studies on Aβ(10–43) fibrils containing disulfide bridges suggested that a β-turn was present from Ser26 to Gly29 (Hilbich et al. 1991). More recently, supported by solid-state NMR data, Petkova et al. (2002) proposed a model for Aβ(1–40) fibrils incorporating a bend region at Gly25–Gly29. This structure would bring the CHC and C terminus into contact, allowing hydrophobic side-chain interactions, an obligate step in fibril formation (Petkova et al. 2002). Our results show that rudimentary forms of the turn or bend structure found in fibril-associated β-sheets are already present in Aβ monomer and are metastable in isolation. An interesting outcome of recent molecular dynamics simulations (Ma and Nussinov 2002) and modeling based on NMR data (Petkova et al. 2002) is the suggestion that the turn or bend region in Aβ fibrils is stabilized by an intramolecular salt bridge involving Asp23 and Lys28. The distance between Cγ of Asp23 and Nζ of Lys28 in the two families of structures (13 Å in family I and 9 Å in family II) would produce a much weaker Coulombic interaction than that associated with the ∼4 Å bond distance in fibrils (Petkova et al. 2002). In addition, in our model, Val24 forms hydrophobic contacts with the side chain of Lys28 and is buried within the core of the turn structure. However, Val24 is solvent-exposed in the model of Aβ(1–40) fibrils proposed by Petkova et al. (2002). This suggests that partial unfolding or rearrangement of the folding nucleus of Aβ monomer occurs during fibril assembly. This rearrangement could be driven by intermolecular interactions, resulting in the formation of an α-helical intermediate (Kirkitadze et al. 2001), which subsequently leads to protofibrils and fibrils (Fig. 6). Experimental studies of fibril assembly thermodynamics, and theoretical treatments thereof, both are consistent with this notion (Kusumoto et al. 1998; Esler et al. 2000a,b; Massi and Straub 2001).