PEDS Advance Access published online on February 13, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm086
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Article |
Different disease-causing mutations in transthyretin trigger the same conformational conversion
Department of Bioengineering, University of Washington, Box 355061, Seattle, WA 98195-5061, USA
3 To whom correspondence should be addressed. E-mail: daggett{at}u.washington.edu
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Abstract |
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Transthyretin (TTR)-containing amyloid fibrils are deposited in cardiac tissue as a natural consequence of aging. A large number of inherited mutations lead to amyloid diseases by accelerating TTR deposition in other organs. Amyloid formation is preceded by a disruption of the quaternary structure of TTR and conformational changes in the monomer. To study conformational changes preceding the formation of amyloid, we performed molecular dynamics simulations of the wild-type monomer, amyloidogenic variants (V30M, L55P, V122I) and a protective variant (T119M) at neutral and low pH. At low pH, the D strand dissociated from the β-sheet to expose the A strand, consistent with experimental studies. In amyloidogenic variants and in the wild-type at low pH, there was a conformational change in the β-sheets into
-sheet via peptide bond flips that was not observed at neutral pH in the wild-type monomer. The same residues participated in conversion in each amyloidogenic variant simulation, originating in the G strand between residues 106 and 109, with accelerated conversion at low pH. The T119M protective variant changed the local conformation of the H strand and suppressed the conversion observed in amyloidogenic variants.
Keywords: amyloidogenic intermediate/molecular dynamics/transthyretin
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Introduction |
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Transthyretin (TTR) is a transporter of the hormone thyroxine in plasma and cerebral spinal fluid (Hagen and Eliot, 1973
; Schreiber et al., 1995). Wild-type TTR and a significant number of variants form amyloid fibrils under physiological conditions causing disease (Saraiva et al., 1984; Cornwell et al., 1988; Westermark et al., 1990; Jacobson et al., 1992, 1997; Saraiva, 2001). In senile systemic amyloidosis (SSA), there is a gradual buildup of wild-type TTR in amyloid fibrils in cardiac tissue (Westermark et al., 1990). This amyloidosis generally leads to a mild disorder affecting an estimated 25% of individuals over 80 years of age. A wide range of mutations accelerates the deposition of amyloid fibrils, mostly in peripheral nerves but also in several organs including heart, kidneys and ocular vitreous (Kelly, 1998). Familial amyloid polyneurpathy is an autosomal-dominant lethal disease commonly associated with point mutations in TTR (Saraiva et al., 1983, 1984).
TTR is a homotetramer that forms a thyroxine-binding channel through its center (Blake et al., 1974, 1978; Wojtczak et al., 1992). Each 127-residue monomer is composed of two four-stranded β-sheets, strands DAGH and CBEF and a short helix in the loop connecting strands E and F (Fig. 1). The monomer dimerizes as two eight-stranded β-sheets, with the two outer H-strands connected by antiparallel β-sheet interactions and buried water molecules. The tetramer is formed by hydrophobic interactions between two dimers through loops connecting strands G to H and strands A to B. TTR is the primary transporter of thyroxine in cerebral spinal fluid and the secondary transporter in plasma. The TTR tetramer also binds retinal-binding protein, the carrier of vitamin A, to protect it from clearance by the kidney (Monaco et al., 1995). The turnover of TTR in plasma is rapid, with a half-life of between 1.5 and 2.5 days (Makover et al., 1988; Divino and Schussler, 1990).
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In many cases, the amyloid assembly process has been studied at acidic pH, where amyloid formation is accelerated due to the role (or presumed role) of endosomes and lysosomes in the process (Glenner et al., 1971
; Cohen et al., 1983; Kelly, 1996). Mouse models expressing human L55P TTR display early aggregates outside the cell and in secretory vesicles in the lumen of the intestine (Sousa et al., 2002). Amyloidosis is transferable between individuals, with liver transplants from FAP patients, leading to TTR deposition in the skin and other organs of the recipient (Sousa et al., 2002). The existence of a slow equilibrium between tetramers and monomers at pH 7 is demonstrated by subunit exchange experiments (Schneider et al., 2001), and amyloid formation occurs at neutral pH for amyloidogenic mutants (Quintas et al., 1997; Lashuel et al., 1998) and wild-type TTR (Quintas et al., 1997, 1999; Lashuel et al., 1998).
There are over 85 identified TTR sequence variants associated with a range of phenotypes (Saraiva, 2001). Although these variants usually have an early onset, as early as an individual’s 20s, the specific pathology is dictated by the mutation. A substitution from Val to Met in position 30 is the most frequent FAP variant in patients, found with high incidence in Portugal, Sweden and Japan (Saraiva et al., 1984). The V122I variant is the most common amyloidogenic mutation worldwide, associated with familial amyloidotic cardiomyopathy in individuals of African descent (Jacobson et al., 1997). It is estimated that 
4% of African Americans are heterozygous for the V122I variant, with an age of onset that is similar to SSA patients at around 60 years of age. One of the most aggressive forms of FAP is associated with a Leu substitution to Pro at residue 55 in β-strand D (Jacobson et al., 1992). Disruptions of hydrogen bonds between strands D and A in the L55P mutant alter the conformation of the outer D strand and contribute to aggregation (Sebastiao et al., 1998; Yang et al., 2003). Although the majority of TTR mutations are associated with amyloidosis, there are other variants described as non-pathogenic. The T119M variant is non-amyloidogenic, and T119M/V30M heterozygotes present a more benign evolution of the disease when compared with V30M kindreds (Alves et al., 1997).
Electron microscopy studies reveal that amyloid deposits from a variety of proteins generally consist of straight or coiled fibrils 7.5–10 nm in width and up to several micrometers in length (Glenner, 1980). Fibrils are characterized by birefrigence under polarized light after straining with Congo Red, and they display X-ray fiber diffraction patterns consistent with a ‘cross-β’ structural model (Glenner, 1980; Jarvis et al., 1993; Fraser et al., 1994). Elementary proto-filaments are proposed to assemble in a hierarchical manner to form higher order fibrils (Shirahama and Cohen, 1967). However, the arrangement of molecules and molecular packing within amyloid fibrils is not fully understood.
It has proved difficult to rationalize the amyloidogenicity of TTR variants because their X-ray crystal structures are so similar, and the subtle structural changes in the vicinity of the mutation are often interpreted as important for amyloidogenesis (Hamilton et al., 1993; Sebastiao et al., 1998, 2001). Recently, our computational studies of the dynamics of amyloidogenic proteins, including wild-type TTR, indicate that such proteins can form an unusual secondary structure, which we dubbed -sheet, and that this conformation may be an intermediate in amyloid formation (Armen et al., 2004a, b). Similar to β-strands, -strands interact to form sheets, but unlike β-sheets, the -sheets are polarized with all of the carbonyl groups from one strand on the same face of the strand and the amide hydrogens on the other face. This conformation leads to a characteristic R/L dihedral angle pattern and alternating residues with positive and negative 
dihedral angles. Although -strands are rare in X-ray crystal structures, they have been observed in the Protein Data Bank (PDB) (Armen et al., 2004a; Daggett, 2006). To investigate the effect of TTR variants on the formation of an amyloidogenic intermediate, and the formation of -sheet, we have performed molecular dynamics (MD) simulations of wild-type TTR monomer and the L55P, V30M, V122I and T119M variants. Here, we describe simulations at neutral pH (pH 6–7), where all His residues are neutral, and at low pH (2 < pH < 4.2) where His, Asp and Glu residues are protonated. We observe the formation of -sheet in amyloidogenic variants and wild type at low pH, with dissociation of the D strand from the β-sheet at low pH. The protective variant, T119M, subverts -sheet formation in the DAGH sheet, which could explain its non-pathogenic phenotype. These simulations complement and extend biophysical studies by providing atomic resolution models for the formation of an amyloidogenic intermediate of disease-causing variants of TTR.
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Methods |
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The X-ray crystal structures of wild-type human TTR and four variants were used as the starting points for MD simulations. The crystal structures correspond to wild-type TTR (1tta 1.7 Å subunit A) (Hamilton et al., 1993
), the L55P variant (5ttr 2.7 Å subunit A) (Sebastiao et al., 1998), the V30M variant (1ttc 1.7 Å subunit A) (Hamilton et al., 1993), the V122I variant (1ttr 1.9 Å subunit A) (Damas et al., 1996) and the T119M variant (1bze 1.8 Å subunit A) (Schormann et al., 1998). The N- and C-termini of truncated structures were modeled on the wild-type structure, and each starting structure consisted of 127 amino acids.
The MD program in lucem molecular mechanics (Beck et al., 2000–2008) was used to simulate the TTR monomers and surrounding water molecules. The protocols and potential energy function have been described previously (Levitt et al., 1995, 1997; Beck and Daggett, 2004; Beck et al., 2005). Simulations of the wild-type protein and each variant were performed for 26 ns (1 ns for thermal equilibration) at 310 K (37°C) at neutral pH (Lys and Arg residues are positively charged, and Asp and Glu residues are negatively charged) and low pH (Asp and Glu residues are neutral, and Lys, Arg and His residues are positively charged). (Also we note that duplicate simulations were performed in all cases and they are in agreement with the results presented here.) The starting structures were minimized for 1000 cycles of conjugate gradient minimization in vacuo to reduce any bad contacts in the crystal structure prior to performing MD. After minimization, water molecules were added around the protein to fill a rectangular box, with walls at least 10 Å away from any protein atom (the number of resulting water molecules ranged from 4902 to 5343). The density of the solvent was set to 0.993 g/cm3 at a temperature of 310 K (Kell, 1967; Haar et al., 1984), and the microcanonical, NVE ensemble was used. Since solvent molecules were explicitly present, no macroscopic dielectric constant was needed, and periodic boundary conditions were used to eliminate boundary effects.
The resulting systems were then prepared for MD. A total of 1000 cycles of conjugate gradient minimization followed by 1000 steps of MD were performed on the water molecules. The water molecules were then minimized again for 1000 cycles, followed by a further minimization of the protein for 1000 cycles. Finally, the entire system was minimized for 1000 cycles. After this preparation, atoms in the systems were assigned velocities according to a Maxwellian distribution. Atoms were allowed to move according to Newton’s equations of motion and the velocities of the atoms were adjusted until the system reached the desired temperature. A 2 fs (2 x 10–15 s) time step was used for both the preparation steps and the full simulation. A 10 Å non-bonded force-shifted cutoff was used and the non-bonded list was updated every three cycles. Figures were created using Chimera (Ferrin et al., 1988).
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Results |
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General features of the trajectories
The conformational behavior of monomeric TTR was simulated using MD, with explicit water at 310 K at neutral and low pH. The C root-mean-square deviations (RMSD) from the crystal structure were measured for residues with clearly discernable electron density in most X-ray crystal structures, from residues 10 to 125 (Hornberg et al., 2000). The RMSD for simulations at neutral and low pH were low and similar in both environments. The lowest RMSD was for the wild-type protein at neutral pH (Fig. 1). The largest average deviations from the crystal structure were in the N- and C-termini, loop regions and in strand D at low pH (Fig. 2).
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The D strand dissociated from the A strand to become a loop in the disease-causing variants and in wild type at low pH (Fig. 3). There was also temporary loss of hydrogen bonding between the D and A strands at neutral pH in wild type, L55P and V30M, although hydrophobic and electrostatic interactions maintained the D strand in position. Hydrogen bonds between the C and B strands, at Ala 45, Gly 37 and Thr 49, were lost at low pH in wild type and L55P for intervals of 2–3 ns. However, the C strand did not dissociate from the β-sheet; instead, it maintained persistent contacts in the hairpin loop between strands B and C, and in the loops connecting strands C to D and A to B. Between strands G and H, there was loss of hydrogen bonding at Thr 119 and Val 121 on the H strand in all simulations except wild type and T119M at neutral pH, leading to exposure of their amide groups.
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The crystal structure of TTR has 11 residues, of which 5 are non-glycine, with positive
angles in each monomer. In our simulations of the wild-type protein at neutral pH, 7 residues retained positive angles and 4 changed to negative values. Six non-glycine residues adopted positive angles in the first nanosecond, and six more later in the simulation. A conversion from β-strand to -strand resulted in an R conformation in one residue and an L conformation (positive and positive 
) in the succeeding residue, with a concurrent rotation of the peptide plane. When the converted -strand interacted with neighboring β-strands, it led to a concerted change allowing the rotated peptide plane to hydrogen bond with the adjacent strands (Fig. 4). Under amyloidogenic conditions, a concerted conformational change occurred in both β-sheets of the monomer, with a larger number of residues participating at low pH. In general, peptide planes of the same residues rotated in each trajectory, and this change usually correlated with a loss of hydrogen bonds to edge β-strands, most frequently strands C and H but also strands A and F. The conversion to -sheet led to a straightening of the β-strands with only a small effect on the C RMSD.
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Conformational changes were more prevalent in simulations of amyloidogenic TTR variants, in particular the V30M protein, and at low pH. The protective variant T119M countered the formation of
-sheet in the DAGH sheet by rotating the peptide plane at Met 119 in the opposite direction to -sheet formed in the amyloidogenic variants. At neutral pH, the wild-type monomer was 1.5–2 Å from the X-ray crystal structure (Fig. 1). However, over the course of the 25 ns simulation, there were transient and isolated conformational changes at several residues that paralleled those at low pH. For example, in the F strand, the peptide plane between His 90 and Ala 91 rotated at 4.36 ns but returned to a native conformation after 900 ps. In the amyloidogenic mutants, transitions in strands C and F were precursors to larger conformational changes.
In the DAGH sheet, the D strand remained hydrogen bonded to the A strand throughout the simulation at neutral pH. Hydrogen bonding between Gly 53 and Leu 55 was lost at 14.5 ns when Leu 55 adopted an L conformation. Subsequently, hydrogen bonds were lost between the A and D strands for short intervals, the longest of 250 ps. Interactions from the loop between strands C and D, and a salt bridge between Glu 54 and Lys 15, maintained the D strand proximal to the A strand (Fig. 3C). In the H strand, the peptide plane between Thr 119 and Ala 120 rotated at 570 ps to direct the carbonyl group toward the solvent. A similar conformation at 119 is observed in the protective T119M variant and at the beginning of the wild type at low pH but not in amyloidogenic mutants. In common with several amyloidogenic simulations, one -strand conversion occurred in strands H, G and A, beginning at 18.6 ns with Tyr 116 and Ser 117 at the bottom of the H-strand. The environment of Tyr 116 is changed in the monomer, since in the TTR tetramer, Tyr 116 is hydrogen bonded to buried water molecules in the dimer interface.
Larger structural changes were observed at low pH, with the D strand dissociating from the DAGH sheet after the first nanosecond of simulation. Under these conditions, Glu 54 in strand D was uncharged and it cannot form a salt bridge with Lys 15, which helps to maintain the D strand in its native position at neutral pH. Leu 55 in strand D adopted an L conformation within the first 100 ps that disrupted hydrogen bonding with strand A. The hydrogen bonding between strands G and H was also disturbed, leading to a rotation of the peptide plane between residues Thr 119 and Ala 120 at 130 ps (Fig. 5A). The carbonyl of Thr 119 was directed away from the β-sheet, similar to simulations of wild type at neutral pH and the protective T119M variant. Unlike these simulations, Thr 119 returned to its native conformation after 2.9 ns.
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At low pH, a concerted conformational change was observed in the DAGH β-sheet to form
-sheet, beginning with the departure of the D strand and a rotated peptide plane in the adjacent A strand, between Lys 15 and Val 16 (Fig. 5A). Over the course of the simulation, -sheet formed between Val 121 and Tyr 116 in strand H, Ile 107 and Leu 110 in strand G and Val 14 and Glu 18 in strand A, with -sheet carbonyl groups directed from strands H to D. In the CBEF sheet, hydrogen bonds with edge strand C, at residues Ala 45 and Gly 47, were lost between 3.4 and 6.4 ns (Fig. 5B). However, the C strand remained attached to the protein core above and below the strand and through interactions in the hairpin loop connecting strands B and C. The loss of hydrogen bonds allowed peptide planes to rotate on the neighboring B strand, between Val 30 and His 31 in strand B and Ser 46 and Gly 47 in strand C (Fig. 5B). A peptide plane flip between His 90 and Ala 91 in the opposite direction, which occurred transiently at neutral pH, suppressed -sheet formation across the CBEF sheet.
Previous NMR experiments on TTR measured the protection of amides protons from exchange with solvent. In these experiments, as the pH was lowered from 5.75 to 4.5, there were changes at well-protected positions in the CBEF sheet, with fewer changes in the DAGH sheet (Liu et al., 2000b). Figure 6A shows the wild-type structure at neutral pH after 25 ns of simulation, colored with the protection factors measured by Liu et al. The CBEF sheet remained native-like at neutral pH, and the large conformational changes indicated by experiments at low pH were not observed. However, hydrogen bonds at Gly 47 and Ser 46 in strand C were frequently lost and reformed, which is reflected by low protection factors for these residues in experiment. In the DAGH sheet, the D strand maintained hydrogen bonds to Val 14 and Val 16, and both valines are well protected in experiment. The loss of hydrogen bonding in the H strand in simulations, at Thr 119 and Val 121, correlates with their lower protection factors. At low pH, the F strand frayed at Ala 91, and strand C became exposed at Ser 46 and Gly 47, both reflected in lower experimental protection factors (Fig. 6B). Although conformational change in the CBEF sheet affected the exposure of amides at Val 30 and His 31, experiments suggest a more widespread change in the CBEF sheet at low pH (Liu et al., 2000b). A larger conformational change would occur if the C strand dissociated from this sheet, allowing a full conversion to -sheet. At edge strands D and H at low pH, the formation of -sheet exposed amides in the H strand and protected the amides of the A strand even when the D strand was far removed from the sheet.
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Amyloidogenic variants
Three amyloidogenic mutants were simulated at low and neutral pH: V30M on strand B is buried in the protein core, V122I on strand H is buried in the dimer interface but exposed in the monomer, and L55P on strand D. All three mutations, independent of pH, displayed the same -strand conformational changes observed in wild-type TTR at low pH.
In the DAGH sheet, three rows of adjacent peptide planes were identified across the β-sheet that underwent conformational change (the ‘rows’ are at the top, center and bottom of the sheet as depicted in the figures). In all cases, the conformational change occurred at the same rate or more rapidly at low pH than at neutral pH. V30M displayed the most rapid conversion, completed in 3 ns, whereas V122I was the slowest, taking up to 17 ns to complete. The central row consisted of the peptide planes between Lys 15 and Val 16 on strand A, Ala 108 and Ala 109 on strand G and Thr 118 and Thr 119 on strand H. In all amyloidogenic variants, except L55P at neutral pH, the peptide plane between Ala 108 and Ala 109 on strand G rotated first, followed by strand A after 10–500 ps, and then strand H between 150 ps and 2 ns later. The order of conversion was different from wild type at low pH, where dissociation of the D strand led to the first rotation on strand A, but similar -sheet resulted.
The lower row, consisting of peptide planes between Met 13 and Val 14, Thr 106 and Ile 107 and Ala 120 and Val 121, converted in all amyloidogenic mutants. This was a concerted conformational change that began at Thr 106 and Ile 107 in strand G and was followed within 100 ps by strand A. In V122I at low pH, the A strand was the first to convert when hydrogen bonding was lost between Thr 106 and Met 13. The peptide plane between Ala 120 and Val 121 on strand H could flip before the G and A strands when hydrogen bonding was lost from the end of the strand. Conformational changes in the upper and central rows were independent events, occurring 1–12 ns apart. In V30M, there was also a conformational change in the upper row of the β-sheet at low pH. This conversion to -strand began on strand H, between Tyr 116 and Ser 117, and propagated to strands G and A. The conformation of strand D differed among the variants. Unlike wild type in which Leu 55 changed into an L conformation, in L55P and V30M at low and neutral pH, Glu 54 adopted an L conformation, and Gly 53 changed from a positive to negative angle. However, in simulations of the V122I variant, neither Glu 54 nor Leu 55 adopted an L conformation.
In the CBEF sheet, all amyloidogenic variants converted to -sheet in a similar conversion as the wild-type protein at low pH. Unlike wild type, in variants at neutral pH, the C strand did not detach from the CBEF sheet. In the V122I variant, -sheet formed across all four strands of the CBEF sheet (Fig. 4A). In other variants, a peptide plane flip between His 90 and Ala 91, observed in wild type at low pH, opposed conversion in the edge strand F and led to reduced hydrogen bonding between the F strand and the sheet around these residues.
In each amyloidogenic variant at low ph, different conformational changes were observed in the CBEF sheet. In the V30M variant, there was -sheet conversion across all strands of the CBEF sheet for >90% of the simulation (Fig. 7). In L55P, the most aggressive variant experimentally, the C strand dissociated from the edge of the CBEF sheet similar to wild type at low pH. However, in both L55P and V122I variants, peptide plane flips between His 90 and Val 93 led to a late propagation of -sheet in the opposite direction to other -sheet conversions (Fig. 4B).
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Two different conformational changes were observed in the CBEF sheet. In all amyloidogenic variants at neutral pH and in V30M at low pH, the changes were identical to the wild type at low pH. In V30M at low pH and V122I at neutral pH, peptide plane flips in strands B and C propagated to adjacent peptide planes in strand E, between Glu 72 and Ile 73, and strand F, between Ala 91 and Glu 92, forming
-sheet across the first sheet. In V122I at low pH, the peptide plane flip between His 90 and Ala 91 in strand F caused a concerted change upon a different set of residues (Fig. 4B), rotating peptide planes between Val 71 and Glu72 on strand E, His 31 and Val 32 on strand B and Ala 45 and Ser 46 on strand A, so that carbonyl groups were in the opposite direction to -sheet in other simulations.
The M119 variant reduces the disease severity associated with other amyloidogenic mutations, in particular V30M. Residue 119 is solvent-exposed on the H strand at the dimer interface, and a larger methionine side chain stabilizes tetrameric TTR. In our simulations, this variant suppressed the formation of -sheet across the DAGH sheet by rotating the peptide plane between Met 119 and Ala 120. At neutral pH, this rotation occurred within 500 ps, and it was followed by adjacent peptide plane flips on strands G and A. This conformational change resulted in an -sheet, with carbonyl groups directed from strand A to H across the sheet, the opposite direction to the -sheet observed in amyloidogenic variants. At 3.5 ns, the upper row of peptide planes in strands A, G and H rotated, as observed in wild-type simulations. A single rotation also occurred in the C, B and F strands, similar to wild-type simulations at low pH.
At low pH, both the peptide planes between Met 119 and Ala 120 and between Val 121 and Val 122 in strand H rotated similar to neutral pH, with their carbonyls directed away from the sheet. At neutral pH, this variant is non-amyloidogenic but at low pH, as in wild-type TTR at low pH, the peptide planes of neighboring A and G strands rotated in the upper and central rows forming -sheet at 9 and 18 ns, respectively. The H strand maintained its conformation and suppressed -sheet in this strand for the remainder of the simulation. In hydrogen exchange experiments on the T119M variant at pH 7 (Liu et al., 2002), low protection factors on the H strand indicate that the protein is in the unconverted tetrameric state (Fig. 6D). There are low protection factors in sheet CBEF at Ala 91, in strand F, and on strand C that suggest that hydrogen bonding in these regions are lost in the tetramer. The simulations suggest that the same loss of hydrogen bonding in the C strand is also found in the monomer (Fig. 6D).
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Discussion |
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Experiment indicates that the monomeric state is the amyloidogenic unit of TTR, and that dissociation of the tetramer leads to a monomeric, amyloidogenic intermediate state (Quintas et al., 2001
). During amyloid formation at low pH, near- and far-UV circular dichroism measurements reflect changes in both secondary and tertiary structure in the intermediate state (Lai et al., 1996; Quintas et al., 1997). The TTR monomer is difficult to study under amyloidogenic conditions since aggregation interferes with most structural techniques. Hydrogen exchange experiments (Liu et al., 2000b, 2002; Olofsson et al., 2004) and protease cleavage sites (Westermark et al., 1990; Kelly et al., 1997) suggest that the C and D strands, at the edge of the β-sheet, separate to expose the A and B strands for aggregation. In MD simulations of the wild-type monomer at low pH, we observed a dissociation of the D strand to form a loop in agreement with experiments (Fig. 3). In simulations of amyloidogenic variants, hydrophobic and electrostatic interactions maintained the D strand close to the A strand even when hydrogen bonds were lost for periods of 1 ns. Unlike the D strand, the C strand did not dissociate from the CBEF sheet although there were time periods when most hydrogen bonds were lost between the B and C strands. Instead, the C strand was anchored by contacts to the hydrophobic core and a structured loop connecting strands B and C.
In combination with gross structural movements, we observed a conformational change in the β-sheets into an -sheet. -Sheet is rare in X-ray crystal structures, but it is observed in MD simulations of amyloidogenic systems (Armen et al., 2004a, b, 2005; Armen and Daggett, 2005), including wild-type TTR, lysozyme variants, β2-microglobulin, the prion protein and polyglutamine. In TTR, -sheet results from the rotation of the peptide plane between two residues in a β-sheet, causing the carbonyl group from the preceding residue to point in the same direction as carbonyl groups of residues on either side. Concurrently, the succeeding residue moves into the L region of the Ramachandran map (Armen et al., 2004a). The rotated peptide groups can hydrogen-bond to adjacent strands, leading to a concerted change in these residues into the same -sheet conformation. Non-glycine residues in the L region are relatively uncommon in the PDB (Lovell et al., 2003), and they are usually important in protein function at evolutionarily conserved positions (Watson and Milner-White, 2002). During the simulation, breathing motions between β-strands lead to reduced hydrogen bonding, allowing residues to assume an L conformation and rotate the peptide plane. This conformational change depends on both a residue’s location in the β-sheet and the type of amino acid. In simulations of TTR variants, Met residues, with long aliphatic side chains, are particularly prone to the rotation of their carbonyl peptide plane, as shown in the T119M suppressor and V30M amyloidogenic variants.
There is considerable experimental evidence for altered tertiary structure in the amyloidogenic intermediate of TTR (Lai et al., 1996; Kelly, 1998; Liu et al., 2000b). As the pH is lowered, there are changes in fluorescence emission and near-UV and far-UV circular dichroism. In the amyloidogenic intermediate, there is a change in the maximum and intensity of the measured far-UV CD signal, but since the spectral properties for an -sheet are not known, interpretation is difficult. Amyloidogenic TTR mutations increase the rate of tetramer dissociation (Quintas et al., 1997; Kelly, 1998), and small molecules that stabilize the quaternary structure slow fibril formation (Miller et al., 2004). Hence, the effect of a mutation on the rate of fibrillogenesis could be influenced more by the rate of tetramer dissociation than the rate of amyloidogenic conversion. As almost all TTR proteins aggregate in the monomeric state, it is difficult to separate a mutation’s effect on the rate of tetramer dissociation from its effects on the rate of conversion.
The conversion to -sheet occurred at the same positions in the β-sheets of different variants, with amyloidogenic variants encouraging conversion and suppressor variants discouraging -sheet propagation. Experimental evidence suggests that conformational changes can take place at these same positions. Previous shorter MD simulations, using a different force field, describe substantial fluctuation in the angle at Ala 120 and Val 121 in the H strand (Yang et al., 2003; Lei et al., 2004). In hydrogen exchange studies of wild-type TTR, the amide of residue Ala120 remains unassigned, whereas those of residues 116 and 118 have broad resonances described by conformational exchange in an otherwise well-ordered region of the crystal structure (Lei et al., 2004). In the crystal structure of a V122 deletion mutant (PDB 1bz8, Schormann et al. to be published), V121 has a positive angle ( = 57, = –174) that directs the carbonyl group into the solvent and prevents wild-type hydrogen bonding to the G strand. In MD simulations, -sheet formation in the DAGH sheet is initiated between Thr 106 and Ala 109 on strand G or between Met 13 and Val 16 on strand A, and these regions contain the most populated -sheet through different simulations (Fig. 7). Peptides from A and G strands of TTR form amyloid in isolation, whereas peptides derived from other parts of the protein do not (Gustavsson et al., 1991; Jaroniec et al., 2002, 2004).
In models of amyloid fibrils, A, B, E, F, G and H strands form the aggregating unit (Serag et al., 2002; Olofsson et al., 2004). Monomers are proposed to associate through the H and G strands, similar to the native dimer, and to form a non-native interface between the A and B strands with a different monomer (Fig. 8A). This model is consistent with hydrogen exchange NMR (Liu et al., 2000b; Olofsson et al., 2004) and EPR experiments, in which paramagnetic probes show the B strands of two subunits are within 8 Å of one another in the amyloid protofibrils (Serag et al., 2002). However, the data are not exclusive to this model, and other arrangements are consistent with hydrogen exchange data, where the surfaces of two sheets are packed in an organized fashion to bring B strands from each into proximity for EPR measurements. Indeed, X-ray diffraction studies on TTR amyloid fibrils suggest that the amyloid protofilament is formed by four β-sheets running parallel to the filament axis, consistent with packing of β-sheets from two TTR monomers (Blake and Serpell, 1996). An -sheet conformation in the amyloidogenic intermediate presents distinct binding surfaces on either edge of the β-sheets. Since different simulations of the CBEF sheet show the carbonyl groups of -sheet can be in the direction of CF or FC, -sheet in CBEF and DAGH could be parallel or antiparallel, with carbonyl groups in the two β-sheets pointing in the same or opposite directions. Neither parallel nor antiparallel -sheets would form amyloid fibrils according to the suggested models, requiring interfaces between like strands, since, in -sheet, the carbonyl groups would be in opposite directions (Fig. 8A), assuming all molecules behave the same way. If the -sheet in strands CBEF and DAGH is antiparallel, subunits could associate either head-to-head (Fig. 8B) or head-to-toe (Fig. 8C) to bring together dissimilar sheets. Conversely, if -sheet is parallel, then association would be possible only head-to-toe. Each arrangement brings into proximity strands from different subunits that are determined to interact in EPR experiments. Magic angle spinning NMR studies on fragments of TTR in mature fibrils are consistent with a β-sheet conformation (Jaroniec et al., 2002, 2004). However, the solid-state NMR data do not exclude -sheet in amyloid fibrils (Armen et al., 2004b), although it is also possible that -sheet could revert to β-sheet in mature fibrils and we hypothesize that -sheet may be more prevalent in the toxic oligomeric intermediates (Daggett, 2006).
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In conclusion, we have performed MD simulations of the TTR monomer and various disease variants at both low and neutral pH. We observed a dissociation of the D strand from the β-sheet that is consistent with experiment, and a reduction of the hydrogen bonding at the C and H strands. We also observed a cooperative conformational change in both sheets of the TTR monomer to form
-sheet. The -sheet formation occurred at the same positions in each simulation and was influenced by amino acid variants and conditions. The T119M protective variant suppresses -sheet formation by changing the local conformation around this residue. -Sheet in the TTR monomer could mediate the orientation and packing of molecules into amyloid protofibrils.
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Footnotes |
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1 Present address: Tessella Plc, 3 Vineyard Chambers, Abingdon, Oxfordshire OX 14 3PX, UK
2 Present address: Department of Molecular Biology, The Scripps Research Institute, 11550 North Torrey Pines Road, La Jolla, CA 92037, USA
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Received December 4, 2007; accepted December 6, 2007.
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