Pathogenic Mutations Induce Partial Structural Changes in the Native

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Pathogenic Mutations Induce Partial Structural Changes in Native #-Sheet Structure of Transthyretin and Accelerate Aggregation Kwang Hun Lim, Anvesh K. R. Dasari, Renze Ma, Ivan Hung, Zhehong Gan, Jeffery W. Kelly, and Michael C Fitzgerald Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00658 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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Biochemistry

Pathogenic Mutations Induce Partial Structural Changes

in

Native

β-Sheet

Structure

of

Transthyretin and Accelerate Aggregation Kwang Hun Lim*,†, Anvesh K. R. Dasari†, Renze Ma , Ivan Hung‡, Zhehong Gan‡, Jeffery W. Kelly§, Michael C. Fitzgerald †

Department of Chemistry, East Carolina University, Greenville, NC 27858, USA. Department

of Chemistry, Duke University, 124 Science Drive, Durham, NC 27708-0346, USA. ‡ Center of Interdisciplinary Magnetic Resonance (CIMAR), National High Magnetic Field Laboratory (NHMFL), 1800 East, Paul Dirac Dr., Tallahassee, FL 32310, USA. § Department of Molecular and Experimental Medicine and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Corresponding author: Kwang Hun Lim, Department of Chemistry, East Carolina University, Greenville, NC 27858, USA. E-mail:[email protected]; (T) 252-328-9805 KEYWORDS TTR, Amyloid, Solid-state NMR, PDSD, amyloidosis, misfolding, V30M, L55P, SPROX, mass spectrometry

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ABBREVIATIONS NMR, nuclear magnetic resonance; PDSD, proton-driven spin-diffusion; TTR, transthyretin; CP, cross-polarization; WT, wild-type; CD, circular dichroism; SPROX, stability of proteins from rates of oxidation; Leu, leucine; Val, Valine; Gly, glycine; Tyr, tyrosine; MAS, magic-angle spinning; HNSB, dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide; GdmCl, guanidinium chloride; H/D exchange, Hydrogen/deuterium (H/D) exchange.


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Abstract Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state NMR structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C-labeling schemes to investigate structural features of βstructured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the

13

C-13C correlation solid-state NMR spectra revealed that WT TTR

aggregates contain an amyloid core consisting of native-like CBEF and DAGH β-sheet structures and the mutant TTR amyloids adopt a similar amyloid core structure with native-like CBEF and AGH β-structures. However, the V30M mutant amyloid was shown to have a different DA βstructure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA β-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures, but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of methionine sidechain also revealed that the sidechain of residue Met-30 pointing between strands D and A is not protected from the oxidation in V30M mutant, while protected in the native state, supporting that the DA β-structure might be disrupted in V30M mutant amyloid.


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Introduction Transthyretin (TTR) is one of the few globular proteins that undergo aberrant conformational transitions from natively folded states into insoluble β-structured aggregates in vivo.1-3 Extracelluar deposition of TTR aggregates in various organs including heart, lung, and peripheral nerves is associated with senile systemic amyloidosis, familial amyloidotic polyneuropathy, familial amyloid cardiomyopathy, and rarely central nervous system selective amyloidosis. 4-6 The TTR amyloidoses exhibit extreme variations of the disease phenotype.4,7-9 For example, aggregation of wild-type (WT) TTR affects primarily the heart and lung, causing senile systemic amyloidosis that affects nearly 25% of the population over age 80.10 Various single-point mutations including V30M and L55P cause exclusively neurological disorders (polyneuropathy). On the other hand, many of the mutations (T60A, I68L, L111M and V122I) are associated with mainly cardiac phenotype, while some of the mutations develop mixed symptoms.11 Such heterogeneity suggests that mutant forms of TTR may have different misfolding pathways and adopt distinct amyloid conformations.12 TTR is a 55 kDa homo-tetrameric protein that binds and transports holoretinol binding protein in the bloodstream. TTR is also the major carrier of thyroxine (T4) in the plasma and cerebrospinal fluid.13 The native state of the 127-residue TTR monomer adopts a primarily βsheet tertiary structure with two four-β-stranded anti-parallel sheets (CBEF and DAGH) that are arranged into a β-sandwich (Fig. 1).14-16 The TTR monomers form a dimer through anti-parallel β-sheet interactions between the H and H’ strands, and between the strands F and F’. Association of two dimers mainly by hydrophobic interactions between the residues in AB and GH loops leads to the formation of tetramer.


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Biochemistry

Figure 1. A schematic diagram of the misfolding and amyloid formation mechanism of TTR.

Previous biophysical studies showed that dissociation of the tetrameric TTR to monomers is a rate-determining step in TTR amyloidogenesis.17-19 The dissociated monomers may then undergo a conformational transition to a partially unfolded intermediate, which self-assembles into amyloid (Figure 1).17-19 It was also shown that most pathogenic single-point mutations destabilize the native TTR tetramer, subsequently facilitating the dissociation of the tetramer and amyloid formation.8,9 However, structural studies using X-ray crystallography revealed that the pathogenic mutations do not induce major changes in the β-sheet tertiary structure that might be responsible for the enhanced aggregation propensity, 20,21 and thereby the precise effect of the mutations on the TTR aggregation remains elusive. The single-point mutations may accelerate only the tetramer dissociation to monomers, and the mutant TTR monomer may have the same misfolding pathway as the WT TTR, which will lead to identical structures of the end product, amyloid. Local structural perturbations by the mutations may also initiate a distinct misfolding transition, resulting in different amyloid structures relative to WT TTR.12 Structural investigation of the soluble precursor states and the final product amyloid derived from WT and mutant forms of TTR would be essential to gain insights into the misfolding pathways. Previous structural studies of TTR aggregates using various biophysical techniques revealed that TTR amyloids contain extensive native-like β-structures. However, the extent of the native-


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like β-structures has been controversial.22-28 Structural investigations of TTR amyloid using electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling suggested that strands C and D dislocate from the main β-sheet structures, exposing strands B and A, respectively, for intermolecular association to amyloid.25,29,30 Hydrogen/deuterium (H/D) exchange NMR experiments on Y114C mutant amyloid also indicated that strands C and D fold away from the β-sheet structure on the basis of relatively lower solvent protection factors of the strands C and D regions.22 However, recent protease digestion experiments suggested a considerably different structure for WT amyloid. It was proposed that strand D in the DAGH βsheet makes contacts with strand E in the amyloid core, implying that the native β-sheet is completely disrupted and rearranged into non-native β-sheet structures in amyloid. 31 Very recently, we reported solid-state NMR structural studies of wild-type (WT) TTR amyloid formed at pH 4.4, revealing that the two native β-sheets (CBEF and DAGH) remain largely unaltered after amyloid formation. 32 We also demonstrated that 13C-13C correlation solidstate NMR experiments with selective

13

CO and

13

Cα labeling schemes are effective for

exploring native-like β-sheet structures in amyloid. 32,33 Here we extended our solid-state NMR structural studies to mutant forms of TTR (V30M and L55P) to investigate the effect of the pathogenic mutations on TTR amyloid structure. Our results reveal that the mutant forms of TTR amyloid have similar native-like structural features (CBEF and AGH) as WT amyloid, which is consistent with our circular dichroism (CD) and mass spectrometry-based thermodynamic analyses of the amyloidogenic precursor states of WT and TTR variants. However, the DA βstructure appears more disordered in the two mutant TTR amyloids, which may be linked to more effective amyloid formation of the mutant TTRs.


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Materials and Methods Protein Expression and Purification Recombinant wild type and mutant forms of TTR were expressed and purified from an Escherichia coli cells transformed from the pMMHa plasmid as described previously.34 Briefly, overexpressed proteins in the soluble fraction of the sonicated lysates were extracted using ammonium sulfate precipitation methods. The precipitates were resuspended with deionized water and purified using anion exchange Q column, followed by size exclusion gel-filtration chromatography using Superdex HR 200 column (GE Health care). The expression and purification methods yielded a large quantity of TTR proteins (~ 80 mg from a 1L M9 medium culture). The protein concentration was calculated using an extinction coefficient of 7.76 × 104 M-1 cm-1 at 280 nm. Amyloid samples were obtained by incubating the protein (0.2 mg/mL) in 200 mM acetate buffer (100 mM KCl, 1 mM EDTA, pH 4.4) for a period of 30 days at 37 °C. The insoluble amyloid was spun down and washed twice with deionized water to remove remaining tetramers and soluble aggregates. The TTR amyloid was examined by transmission electron microscopy (TEM) and thioflavin T (ThT) binding assay in our previous studies.33

CD spectroscopy The CD spectra were recorded by scanning from 250 nm to 190 nm on a Jasco J-815 spectropolarimeter (Easton, MD) using 1 and 3 mm path length Suprasil quartz cells depending on the protein concentration. Very low protein concentrations of 0.09 and 0.02 mg/ml in 15 mM sodium acetate buffer (pH 4.4) were used for the CD experiments.


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Solid-state NMR Solid-state NMR spectra were recorded using Bruker 600- and 830-MHz spectrometers equipped with a 3.2 mm MAS probe. Two-dimensional

13

C-13C correlation NMR spectra were

recorded using a proton-driven spin diffusion (PDSD) mixing scheme at spinning frequencies of 17 kHz (600 MHz) and 11 – 12 kHz (830 MHz). The spinning speed was set close to the ∆ωiso = ωr (600 MHz) and ∆ωiso = 2ωr rotational resonance (RR) conditions 35,36 for efficient polarization transfer. Since the second spinning sideband from 13CO carbons is still observable under the RR conditions, the spinning speed was set slightly below for each 13CO/13Cα spin pairs such that the sideband of the carbonyl carbon appears ~ 5 ppm away from the NMR resonance of the carbon. The 90° pulse-lengths for 1H and

13

13

Cα

C were 3.0 and 2.5 µs, respectively, and the two-

pulse phase-modulated (TPPM) decoupling scheme was employed with a radio-frequency field strength of 85 kHz. For the 2D PDSD spectra, complex data points of 1024 × 362 and 1024 × 145 were collected for the native and amyloid states, respectively, with an acquisition delay of 2 sec, and 48 – 64 FIDs were accumulated for each t1 data point. The cross-peaks observed in all of the WT PDSD spectra were assigned in our previous solid-state NMR study.32 For the selectively

13

CO- and

13

Cα-labeled samples, the M9 media supplemented with

unlabeled amino acids (100 mg per liter culture) and

13

CO- and

13

Cα-labeled amino acids (50

mg/L) were used for protein expression. In our bacterial expression system, serine was synthesized from glycine, and thus 13Cα-Gly was used to label serine. The selective labeling of the specific amino acid was confirmed by

13

C solid-state NMR spectra of the native state of

TTR. For more extensive amino acid selective labeling, 15N-NH4Cl and 13C-glucose were used in M9 media supplemented with the other unlabeled amino acids (100 mg/L) by utilizing biosynthetic pathways of the amino acids in bacteria. 37 For example, in order to selectively label


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eleven amino acids (P, L, M, V, K, D, A, G, S, H and T), M9 medium was supplemented with 15

N-NH4Cl, 13C-glucose and the other nine unlabeled amino acids (100 mg/L).

Chemical Modification and Mass Spectrometry-based Analyses The chemical denaturation curves generated here were obtained using a previously established tryptophan

modification

protocol

involving

dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium

bromide (HNSB). 38 Briefly, the HNSB experiments were performed by reacting the protein (1 µM) with HNSB (3.68mM) in a series of different buffers (pH 4.4) that contained increasing concentration of guanidinium chloride (GdmCl). The HNSB modification reactions in each buffer were allowed to proceed for 10 min at 37 °C. The reactions were quenched during a desalting step using Millipore C18 Zip-Tips. The desalted protein samples were each analyzed by MALDI-TOF mass spectrometry using sinapinic acid as the matrix. The MALDI mass spectra were acquired on an UltraFlex II TOF/TOF (Bruker Daltonics) mass spectrometer in the reflective and positive ion mode using a smartbeam Nd:YAG laser (355 nm). Spectra were collected using the following instrument parameters: 25kV ion source 1 voltage, 21.9kV ion source 2 voltage, 9.9kV lens voltage, 26.3kV reflector voltage, 13.7kV reflector 2 voltage, 100ns pulsed ion extraction, and matrix gating to 4000 Da. The protein ion signal in the resulting mass spectra were used to extract ∆Masswt,av values as we have previously described. 38 The time course studies of Met-30 oxidation in V30M TTR were performed by reacting the protein (1 µM) with H2O2 (100 mM) at pH 4.4 (200 mM acetate buffer) or pH 7.4 (200 mM phosphate buffer) for a specific of time at 37 °C. The reaction was quenched by adding a 6-fold molar excess of L-methionine over H2O2. A buffer exchange was performed using Amicon Ultra 10K centrifugal filters to obtain protein solutions in 100 mM triethyammonium bicarbonate


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buffer (pH 8.5). The protein solutions were digested with Lys-C protease, and the resulting peptide solutions were analyzed by MALDI-TOF mass spectrometry using α-cyano-4hydroxycinnamic acid (HCCA) as the matrix for MALDI-MS analysis. For determination of the oxidation status of Met-30 in the V30M mutant, the MALDI analysis was focused on the ion signals from the Met-30 containing peptide VLDAVRGSPAINVAMHVFRK in both its wildtype and oxidized forms. The MALDI ion signals of the wild-type and oxidized forms of the VLDAVRGSPAINVAMHVFRK peptide were identified by their masses. MALDI mass spectra for the time-course experiment were acquired on a Voyager-DE PRO (Applied Biosystems) mass spectrometer in the linear and positive ion mode using a nitrogen laser (337 nm). Spectra were collected using the following instrument parameters: manual control mode, 20kV accelerating voltage 93% grid, 0.05% guide wire, 100 nsec delay time, 60 shots/spectrum, and low mass gate 500 Da. The time-course data were analyzed as previously described. 38 Briefly, the increases in mass as a function of reaction times were obtained by calculating ∆Masswt,av values, using a weighted average of the intensities of the ion signals from the wild-type and oxidized peptide, and by fitting the data to a single exponential equation.

Results β-structured amyloidogenic precursor states The native state of TTR monomer adopts a primarily β-sheet tertiary structure, in which eight β-strands denoted A-H are arranged into a β-sandwich with two β-sheets (CBEF and DAGH, Figure 1). Thus, a local unfolding transition of the outer β-strands may be able to trigger aggregations to cross-β structured amyloid containing substantial native-like structures. Circular


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dichroism (CD) spectroscopy was used to examine structural features of amyloidogenic precursor states of WT and mutant forms of TTR (V30M and L55P) at the most amyloidogenic pH of 4.4. Our previous CD analysis of WT TTR showed that the amyloidogenic precursor state of WT TTR has almost identical secondary structures to those of native state of TTR. 33 The structural feature of the WT amyloidogenic state was compared to those of the TTR variants using CD spectroscopy (Figure 2). Notably, the amyloidogenic states of the more aggressive TTR variants exhibit similar CD spectra with the same minimum signal intensity at 212 nm, indicative of β-sheet conformations. The CD spectra suggest that the WT and TTR variants share similar native-like secondary structures.

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ellipticity (mdeg)

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Biochemistry

Figure 2. CD spectra of WT and two mutant WT V30M L55P

10 5

forms of TTR (0.09 mg/ml) in 10 mM sodium acetate buffer (pH 4.4) recorded at 20

0

o

C.

Identical spectra were obtained at a lower protein

-5

concentration (0.02 mg/ml), suggesting that the

-10 190

200

210

220

230

240

wavelength (nm)

250 CD signals originate mainly from monomeric

precursor states of TTR. Four scans were collected and averaged for the CD spectra.

It was shown that TTR tetramers are dissociated to monomers at the amyloidogenic condition of pH 4.4. Previous ultracentrifugation experiments showed that tetrameric TTR is in a dynamic equilibrium with monomeric forms of TTR at pH 4.4 (approximately 25 % of the tetramers are present at a protein concentration of 0.2 mg/ml).24 Thus TTR would exist predominantly as a


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monomeric form under our experimental conditions (0.09 – 0.02 mg/ml), which indicates that the CD signals recorded at pH 4.4 mostly originate from amyloidogenic TTR monomers. Thermodynamic stabilities of the amyloidogenic precursor states were examined using a chemical modification and mass spectrometry-based approach, which has been previously described. 38 In this mass spectrometry-based analysis, we monitored the unfolding transition of the precursor states by evaluating the chemical denaturant dependence of the reaction of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNSB) with tryptophan (Trp) residues.38 At increasing concentrations of chemical denaturant, the TTR protein is unfolded and the Trp residues are more exposed to solvent and more readily react with HNSB, resulting in an increased mass of the protein that can be detected by using mass spectrometry. In the natively folded state of TTR, both Trp residues (W41 and W79) appear to be largely protected from the reaction with HNSB (Supplemental Figure S1), as evidenced by the relatively low ∆Masswt,av values of the pre-transition baselines of the chemical denaturation curves in Figure 3. In the presence of increasing concentrations of chemical denaturant (GdmCl in this work), the protected Trp residues in the native state become available for the chemical reaction with HNSB, leading to an increase in the protein mass, as demonstrated in Figure 3. Thus, the chemical denaturant dependence of the HNSB modification reaction can report on the thermodynamic stability of the WT and mutant forms of TTR. The cooperative unfolding transitions observed in Figure 3 suggest that the amyloidogenic precursor states adopt folded conformations. A qualitative analysis of the transition midpoints of the chemical denaturation curves generated in Figure 3 also indicates that the precursor states of the TTR variants are less stable than that of WT TTR. The less stable amyloidogenic precursor states of the TTR variants might be linked to the stronger aggregation propensity of the TTR variants.


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Biochemistry

Figure 3. Chemical denaturation curves generated for WT, V30M, and L55P TTR using the tryptophan modification protocol. The vertical dotted lines indicate the concentrations of guanidinium chloride (GdmCl) at the transitions midpoints, which were 3.8, 1.6 and 1.4 M for WT, V30M, and L55P, respectively. The data points and error bars represent the average and standard deviation of the ∆Masswt,av values determined from the 8 mass spectra collected on each sample.

Structural features of the amyloid states derived from WT and mutant forms of TTR (V30M and L55P) The CD and mass spectrometry data described above reveal that the amyloidogenic precursor states of WT and mutant forms of TTR adopt overall very similar folded native-like conformations. TTR effectively forms amyloid at pH 4.4 via a downhill mechanism where major conformational changes are not necessary.39 Thus, the native-like structural features observed in the CD and mass spectrometry-based analyses may be retained in TTR aggregates. Solid-state NMR was used to probe the native-like β-structures in amyloid state of WT TTR. Twodimensional (2D) cross-polarization (CP) based

13

C-13C and

15

N-13C correlation NMR spectra


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were acquired for amyloid state of WT TTR and compared to that of the native state (Supplemental Figure S2). For the 13C-13C correlation experiments, proton-driven spin diffusion (PDSD) mixing scheme40 with a short mixing time (25 ms) was used to record intra-residue correlation NMR spectra (Supplemental Figure S2a and S2b). The

15

N-13C correlation NMR

spectra shown in Supplemental Figure S2c were acquired for the amide 15N and the 13Cα (NCA). The 2D 13C-13C correlation and NCA spectra show that the NMR resonances from the amyloid state (red) are overlapped well with those of the native state (black), suggesting the presence of native-like structures in amyloid state. It is notable that some of the NMR resonances in the native state are not observed in the amyloid state spectrum. Previous solid-state NMR studies of amyloids derived from various polypeptides revealed that strong signals in the dipolar-based NMR spectrum of amyloid mostly originate from highly structured amyloid core regions, while NMR signals are not observable from other unstructured regions due to extensive linebroadening.41-44 Our previous structural analyses of WT TTR amyloid also showed that loop regions such as AB and EF loop are more disordered, 33 suggesting that NMR signals from the disordered regions are not observable in the 2D solid-state NMR spectrum for the amyloid state of WT TTR in Supplemental Figure S2. The solid-state NMR studies were extended to investigate structural features of mutant forms of TTR amyloid. Figure 4a shows

13

C cross-polarization (CP) magic angle spinning (MAS)

NMR spectra of amyloid states of WT and TTR variants (V30M and L55P). The three amyloid states exhibit a similar pattern in the chemical shifts, while NMR signals at aliphatic regions (20 – 70 ppm) are weaker in intensity for the two mutant forms of TTR amyloid. These NMR results indicate that amyloid states of the two TTR variants might be slightly more disordered than that of WT TTR. Structural features of the three amyloids were also examined with 2D

13

C-13C


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correlation NMR experiments (Figure 4b). Overlay of the aliphatic regions of the 2D spectra for the three amyloid state suggest that the amyloid state of WT and mutant forms of TTR share a similar structural feature in amyloid core regions that produce strong NMR signals in the dipolarbased 2D correlation experiments.

Figure 4. (a)

13

C CPMAS spectra of the amyloid states of WT (black), V30M (red), L55P

(green) TTR where eleven amino acids (P, L, M, V, K, D, A, G, S, H and T) are uniformly 13

C/15N labeled. (b) Overlay of the 2D

13

C-13C correlation NMR spectra of the three amyloid

states with a PDSD mixing time of 25 ms. Each PDSD spectrum was compared with the other two PDSD spectra separately [WT (black), V30M (red), L55P (green)].


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DAGH β -sheet structure in amyloid states The combined structural analyses described above indicate that the amyloid sates of TTR contain extensive native-like β-sheet structures. Solid-state NMR has proven to be a powerful tool for detailed structural characterization of non-crystalline amyloid at atomic resolution. 43-55 However, structural analyses of the tertiary β-structure within this 127-residue TTR amyloid are a daunting task. We recently demonstrated that specific labeling schemes, which generate isolated 13CO-13Cα dipolar-coupled spin pairs in the native β-sheets (Figure 5a), are of great use for probing the native β-sheet structure in amyloid state.32,33 For example, selective

13

CO-Leu

and 13Cα-Tyr labeling will produce three dipolar-coupled 13CO-13Cα spin pairs at distances of 4– 6 Å in the AGH strands. Solid-state NMR experiments that detect spin-pairs with a separation of up to 6 Å can then be used to investigate any structural changes in the β-structure. Figure 5b shows 13C cross-polarization (CP) magic-angle spinning (MAS) NMR spectra of the native and amyloid states of TTR. The NMR peaks at ~175 and ~60 ppm in the 13C CPMAS spectrum of the L/Y labeled TTR confirms the selective enrichment of

13

CO and

13

Cα carbons, respectively

(Figure 5b and Supplemental Figure S3). Although the NMR resonances in the amyloid states are substantially broader than those of the native state, our previous solid-state NMR experiments showed that TTR amyloid used in our studies is a uniform protein assembly consisting of a single monomeric core conformer on the basis of a single set of cross-peaks in extensive

13

C-13C correlation solid-state NMR spectra.

32,33

It is also notable that the NMR

resonance for residue Y78 in EF loop is more significantly broadened in the amyloid states, indicating that the loop regions might be disordered in the amyloid states.


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a

C

B

K48 G47

A29

E

F

V30 5.0 I73 4.9 A91 5.0Å S46 H31 E72 E92 V32 A45 V71 V93 K70 4.9 V94 F44 F33 P43 R34 4.4 Y69 F95 E42 K35 I68 T96 W41 A36 G67 A97

A D

G 5.7 R104

H T123

L12 Y105 5.0 V122 4.7Å M13 T106 V121 L55 V14 4.3 I107 A120 E54

K15 V16 L17 D18

A108 A109

T119 4.3 T118 S117 L110

L111

Y116

S112 5.2 S115

b

Figure 5. (a) Selective labeling schemes for the solid-state NMR experiments. The (green) and

13

13

CO

Cα (red) carbons of the amino acids are labeled if the internuclear distance in the

native TTR tetramer is within 6 Å. (b)

13

C CPMAS spectra of the native and amyloid states of

TTR prepared from 13CO-Leu and 13Cα-Tyr labeled protein. The NMR resonance of the residue Y78 in the EF helix is slightly shifted upfield and becomes much broader in amyloid state, suggesting that the EF helix becomes disordered in the amyloid state.

The 2D 13C-13C correlation experiments using a long PDSD mixing time of 500 ms were used to probe the weakly dipolar-coupled spin pairs at distances of 5 – 6 Å in AGH β-structure in the amyloid states of WT and TTR variants (Figure 5a and Figure 6). Figure 6a (the left spectrum) shows overlay of the PDSD spectra for the L/Y (black) and L/M (red) spin pairs in WT amyloid state. The two cross-peaks for the L110-Y116 and L111-Y116, and one cross-peak for L12-Y105 (black in Figure 6a) were observed in the WT TTR amyloid. The 2D NMR experiments were


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also conducted to probe the L/M spin pair in the DA β-structure for WT amyloid (Figure 5a and red in the left spectrum in Figure 6a). The two cross-peaks for the L55-M13 and L12-M13 were detected in the WT amyloid state. These NMR results indicate the presence of native-like DAGH β-structure in WT amyloid state. Additional evidence for the DAGH β-structure was obtained with the 2D PDSD experiments on a

13

CO-Val/13Cα-Tyr labeled TTR amyloid (Figure 5a and

right spectrum in Figure 6a) that supports a close proximity between strands G and H. The solid-state NMR experiments were conducted on amyloid states of the two TTR variants (Figure 6b and 6c). The NMR cross-peaks for the L/Y and V/Y spin pairs in the AGH β-structure (black in Figure 6) were all observed in the mutant forms of TTR amyloid, suggesting that the mutant amyloids also contain the native-like AGH β-structure. Similar structural features in AGH structure is supported by additional 2D experiments on I/V spin pairs (AG strands in Figure 5a and Supplemental Figure S4). In contrast to the spin-pairs in the AGH β-structure, the cross-peak from the L55-M13 spin pair in DA β-structure is not observed in V30M amyloid (Figure 6b). The different 2D spectrum for V30M mutant indicates that the V30M TTR may have a different DA substructure in amyloid state. In the stronger amyloidogenic mutant L55P, the distance between P55 and M13 is higher (6.5 Å) even in the native state, in comparison to the native tetrameric TTR (5 Å), as shown in Supplemental Figure S5. The strand D is also more disordered in the native state of the more amyloidogenic mutant L55P TTR. Theses results indicate that destabilization of the DA β-structure may be critical to the effective amyloid formation, as suggested by previous computational studies. 56,57


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Figure 6. 2D PDSD spectra of amyloid states of the 13CO-Leu/13Cα-Tyr (black, left; labeled on AGH strands),

13

CO-Leu/13Cα-Met (red; labeled on DA), and

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CO-Val/13Cα-Tyr (black, right;

labeled on GH) TTR with a contour level of 3.0 % with respect to the diagonal peak obtained using a mixing time of 500 ms at a 1H frequency of 830 MHz. L/Y and L/M spin pairs were unambiguously assigned in our previous solid-state NMR studies.32 In order to confirm that the cross-peaks originate from the spin pairs within the TTR monomer, the 2D PDSD experiments were conducted on the mixture of singly 13CO- and 13Cα- labeled TTRs (1:1 ratio, for example a mixture of

13

CO-Leu-TTR and

13

Cα-Tyr-TTR) and no cross-peak was observed from the

mixture.


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Native-like CBEF β -sheet structure The solid-state NMR experiments were extended to probe CBEF β-structure. Our previous solid-state NMR experiments showed that cross-peaks for I73-M30 and I73-A91 spin pairs in the BEF β-structure (Figure 5a) were observed in the 2D PDSD spectra obtained for V30M amyloid,32 which is in good agreement with the NMR spectra for the I/V labeling scheme (Supplemental Figure S4). Additional F/Y spin pairs in the BEF β-structure were investigated with the

13

C-13C correlation experiments (Figure 7). Our recent solid-state NMR experiments

combined with single-point mutagenesis successfully assigned the two NMR cross-peaks for F33-Y69 and F95-Y69 that are overlapped in the 2D spectrum of WT TTR amyloid sample.32 The two cross-peaks for the F33-Y69 and F95-Y69 observed in the WT amyloid (Figure 7) were also detected with the same line width and intensity in those of the two mutant forms, suggesting the two single-point pathogenic mutations do not affect the BEF β-structure in mutant amyloids.

Figure 7. 2D PDSD spectra of amyloid states of the

13

CO-Phe and

13

Cα-Tyr (labeled on BEF

strands) TTR obtained using a mixing time of 500 ms at a 1H frequency of 830 MHz. * denotes spinning sidebands.

The 2D PDSD experiments were also conducted to investigate CB β-structure in the amyloid states of the TTR variants (Figure 5a and Figure 8). For the structural study of the CB


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substructure, three amino acids (13CO-His,

13

Cα-Gly and

13

Cα-Ser) were selectively labeled,

generating the H31-S46 spin pair in strands B and C, respectively (Figure 8). The 2D

13

C-13C

correlation NMR spectra were collected for the amyloid states of the two TTR variants and compared to that of WT TTR amyloid. The two cross-peaks in the WT amyloid PDSD spectrum were assigned in our previous solid-state NMR studies.32 The presence of the similar cross-peak for H31-S46 in both V30M and L55P amyloid suggest that the two mutant amyloids have a similar CB substructure. Additional evidence for the native-like CB β-structure in the amyloid states was obtained with the 2D PDSD experiments of

13

CO-Gly and

13

Cα-Val TTR samples

(Figure 5a and 9). The cross-peak from the G47-V30 spin pair that is absent in the V30M spectrum supports the intact CB β-structure in the WT and L55P mutant amyloids.

Figure 8. 2D PDSD spectra of amyloid states of the 13CO-His, 13Cα-Gly, and 13Cα-Ser (labeled on CB strands) TTR obtained using a mixing time of 500 ms at a 1H frequency of 830 MHz. * denotes spinning sidebands, and the same contour level was used for the three spectra.


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Figure 9. 2D PDSD spectra of amyloid states of the 13COGly and

13

Cα-Val (labeled on CB strands) TTR using a

mixing time of 500 ms at a 1H frequency of 830 MHz [WT (black), L55P (green), and V30M (red)]. The cross-peak marked by ? could not be assigned. The cross-peak indicated by the dashed line is absent in the V30M amyloid (red), and thus it was assigned as G47-V30.

Time-course analysis of Met-30 oxidation in V30M TTR The solid-state NMR experiments of V30M amyloid described above suggest that strand D might be unfolded in amyloid state, which may fully expose strand A for intermolecular interactions. The sidechain of residue Met-30 points toward the DA substructure (Supplemental Figure S6) in the native V30M TTR, and the longer sidechain of methionine than that of valine may destabilize the DA structure, inducing a structural change under the amyloidogenic condition. In order to probe the local environment of the methionine sidechain, oxidation rates of the methionine sidechain were analyzed using mass spectrometry. 58 In this mass spectrometrybased approach we measured the time-course of the H2O2 oxidation of the Met-30 sidechain. In the native tetrameric V30M TTR at pH 7.4, the sidechain of residue Met-30 is protected from the oxidation (Figure 10). On the contrary, the Met-30 sidechain is readily oxidized at pH 4.4, suggesting that the M30 sidechain protected in the native state is exposed to solvent in the amyloidogenic precursor state at pH 4.4. We note that the H2O2 oxidation of an unprotected Met


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residue is not significantly different at pHs 4.5 and 7.4 (see Supplemental Table S1). Thus, the different reaction rates observed in Figure 10 appear to be due to structural differences in the protein. We also note that the second-order reaction rate constant calculated from the time-course data on V30M TTR at pH 4.4 and 37 oC (63 M-1h-1) was only slightly smaller than that measured for an unprotected methionine in the model peptide under the identical condition (82 M-1h-1)59 (see Supplemental Table S1).

Figure 10. Time course analysis of Met-30 sidechain oxidation with H2O2 in V30M TTR. Data obtained on the Lys-C generated TTR peptide VLDAVRGSPAINVAMHVFRK, which contains residue Met-30, are shown. The data points and error bars represent the average and standard deviation of the ∆Masswt,av values determined from the five mass spectra collected on each sample. The dotted blue line represents the best fit of the pH 7.4 data to a line. The solid red line represents the best fit of the data to a single exponential equation.


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Discussion Despite the extensive mechanistic studies of TTR misfolding and amyloid formation, detailed molecular mechanism has remained largely unknown. Structural characterizations of amyloidogenic precursor states and the final product amyloid are essential to the understanding of the amyloid formation mechanism. However, structural studies of amyloidogenic precursor states are of great challenge due to their dynamic, aggregation-prone properties. In this study, we employed CD spectroscopy and mass spectrometry to investigate structural features of amyloidogenic precursor states of TTR using a very low protein concentration (1 µM) to minimize aggregation. Our structural analyses using CD spectroscopy showed that the amyloidogenic precursor states of WT TTR adopt a native-like β-sheet structure at the most amyloidogenic pH of 4.4. The CD results are in good agreements with our mass spectrometrybased thermodynamic analyses of WT TTR at pH 4.4 that showed the precursor state undergoes a cooperative unfolding transition upon additions of a denaturant, characteristic of folded proteins. The CD spectra, in conjunction with mass spectrometry-based unfolding studies, clearly demonstrated that amyloidogenic precursor states of WT TTR adopt largely folded native-like βsheet conformations. TTR amyloidogenesis is characterized by more than 100 single-point mutations that cause early onset familial amyloidoses. Most of the pathogenic mutations tested in vitro were shown to kinetically or thermodynamically destabilize the native tetrameric state of TTR, accelerating the dissociation of the tetramers to monomers, which is the rate-determining step in TTR amyloid formation process. 8,9,16,24 However, the effect of the single-point pathogenic mutations on the molecular structure of the monomeric precursor states and amyloid has not been reported. Previous structural studies using X-ray crystallography revealed that the pathogenic TTR


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variants have almost identical crystal structures to that of the tetrameric state of WT TTR. Only local structural perturbations at the vicinity of the mutation were observed. Recent relaxation NMR experiments identified flexible regions undergoing conformational fluctuations that might be linked to the dissociation of the TTR tetramer to aggregation-prone monomers.60 The pathogenic mutations (V30M and L55P) were shown to induce more extensive conformational fluctuations of the same flexible regions as those observed for WT TTR. However, a notable difference was observed in strand D. The short strand with little flexibility in WT TTR undergoes substantial conformational fluctuations in the pathogenic mutants.60 These NMR results are largely consistent with our biophysical studies that showed the amyloidogenic states of the TTR variants are less stable than that of WT TTR. In addition, our solid-state NMR results reveal that amyloid states of WT and two mutant forms of TTR adopt a similar native-like CBEF and AGH β-sheet structure, but different DA substructures were observed in the mutant amyloids. The solid-state NMR results described here suggest that the native-like DA substructure is maintained in the WT amyloid state on the basis of the cross-peak from the L55/M13 spin pair. However, the NMR signal for the spin pair in the DA β-strands was not observed in V30M amyloid, indicating that strand D may undergo a structural change. Although the single-point mutation (V30M) is observed in strand C, the side chain of the methionine points toward the strands D and A in the other DAGH β-sheet (Supplemental Figure S6). The longer side chain of methionine than that of valine may crash with other side chains in strands D and A, inducing a structural change in the DA substructure, as was suggested by the previous relaxation dispersion NMR experiments. 60 In the stronger amyloidogenic mutant L55P, the distance between residues P55 and M13 is higher (6.5 Å) even in the native state, in comparison to the native WT


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tetrameric TTR (5 Å), as shown in Supplementary Figure S5. The strand D is also more disordered in the more amyloidogenic mutant L55P TTR, which is consistent with our mass spectra-based analysis that showed the amyloidogenic state of the mutant is less stable than that of WT TTR. In addition, the sidechain of residue Met-30 that points toward DA substructure is protected from oxidations in the native V30M, while the sidechain is readily available for the oxidation in the amyloidogenic state of V30M TTR. The combined results indicate that the DA substructure becomes disrupted in the amyloidogenic states of the TTR variants. Previous H/D exchange experiments also reported the structural change of strand D in another TTR variant (Y114C) amyloid. 22 In addition, triple mutations on strand D (G53S/E54D/L55S) were shown to slightly unfold the strand D, accelerating the aggregation.61 Taken all together, the destabilization of the DA β-structure under amyloidogenic conditions may trigger more effective amyloid formation of the TTR variants, as suggested by other biophysical studies. 22,34,56,62 The structural studies of the amyloidogenic precursor states and the final product, amyloid, provide valuable insights into TTR aggregation pathway. Our previous solid-state studies revealed that the native-like CBEF and DAGH β-structure is retained in WT TTR amyloid state, while AB loop regions that are hydrogen-bonded with strand A undergo a structural transition. 33 The partly exposed strand A might be involved in intermolecular interactions essential to TTR amyloid formation. In our present solid-state NMR studies reported here, the amyloid states of the two TTR variants are also shown to adopt the similar native-like CBEF and AGH β-sheet structure. However, strand D appears undergo a structural change in the amyloidogenic states of the TTR variants, which may fully expose strand A for intermolecular interactions. The structural change of the strand D may accelerate amyloid formation of the pathogenic TTR variants, which is in good agreements with previous computational studies. 56,62


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The structural information of the monomeric conformer in amyloid state will also help to develop a quaternary structural model of TTR amyloid. The combined solid-state NMR studies with CD and mass spectrometry-based analyses show that the monomeric conformer in WT TTR amyloid adopts a native-like β-sheet structure, suggesting that only outer strands (C, F, D, H, and part of the strand A due to the structural change of the AB loop region) are available for the intermolecular interactions. In addition, previous relaxation dispersion NMR experiments of the monomeric and tetrameric TTR showed that CBEF β-sheet remains stable while the other βsheet (DAGH) undergoing extensive conformational fluctuations on millisecond time scales associated with misfolding and amyloid formation. 60,63 These NMR results suggest that DAGH β-sheet may be involved in intermolecular associations. Indeed, recent mechanistic studies of TTR aggregation showed that intermolecular interactions between strands H play a key role in TTR amyloid formation, and strand A has a strong aggregation propensity. 64 In addition, TTR dimers cross-linked between the strands H and H’ were shown to form amyloid. 65 On the basis of the previous biophysical studies and our solid-state NMR results, we propose that the nativelike monomeric TTR forms a dimer through H-H’ interactions, and the dimers may oligomerize via A-A’ interactions (Figure 11), as was suggested by previous EPR studies.25 The more exposed strand A due to the structural change of strand D in the TTR variants may not only facilitate the A-A’ interactions, accelerating TTR aggregation, but also lead to different intermolecular association patterns and thus distinct quaternary structures. The mutant amyloids with distinct quaternary structures may interfere with tissue-specific cellular components such as receptors and signaling proteins, resulting in phenotype diversities observed in TTR amyloidogenesis.


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Figure 11. Proposed structural models for the amyloid states of WT and mutant forms of TTR. On the basis of our solution and solid-state NMR results, we propose that the CBEF β-sheet remains unchanged in the amyloid states. However, we cannot completely rule out that the TTR amyloid used in this work may contain a minor conformer where the strand C is unfolded and involved in intermolecular interactions, as was proposed by the EPR studies based on the EPR signal from strands B and B’.25 It is also plausible that the EPR signal may come from interactions between protofibrils that contain native-like CBEF β-sheet structures.

Finally, there is growing evidence that amyloid formation of natively folded proteins can proceed via native-like intermediates populated by conformational fluctuations of the natively folded state. 66-69 Although structural characterization of the amyloidogenic intermediate state is essential to understanding molecular mechanism of the amyloid formation process, the structural analysis is of great challenge due to transient and aggregation-prone nature of the intermediate states. Our CD and mass spectrometry-based thermodynamic analyses used relatively low protein concentrations (~ 1 µM, monomer concentration) to prevent protein aggregation, which allowed the structural characterization of the amyloidogenic precursor states. Solid-state NMR was then used to investigate the native-like structures in the final product, amyloid, using the selectively labeling schemes. The combined structural studies of the initial and final states of the protein in amyloid formation process would provide valuable insights into molecular


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mechanisms of misfolding and amyloid formation of the other natively folded proteins such as superoxide dismutase 1 (SOD1) and β-microglobulin.

ASSOCIATED CONTENT Supporting Information. 2D PDSD NMR spectra for the mutant TTR amyloids. Schematic diagrams of monomeric TTR displaying sidechains of Trp and Met. Oxidation rates for the Metcontaining model peptides. The following files are available free of charge.

AUTHOR INFORMATION Corresponding Author Email: [email protected] Funding Sources This work was supported by NIH Grants NS084138 (KHL), NS097490 (KHL) and DK46335 (JWK), and the Skaggs Institute of Chemical Biology (JWK). The solid-state NMR spectra were acquired at the National High Magnetic Field Laboratory, which is supported by NSF Cooperative Agreement No. DMR-1157490 and the State of Florida. Notes The authors declare no competing financial interests.


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ACKNOWLEDGMENT We thank Profs. P. E. Wright (Scripps) and D. E. Wemmer (University of California at Berkeley) for helpful discussion. We also acknowledge Prof. Kenney (ECU) for assistance in the CD experiments.

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Pathogenic Mutations Induce Partial Structural Changes

in

Native

β-Sheet

Structure

of

Transthyretin and Accelerate Aggregation Kwang Hun Lim*,†, Anvesh K. R. Dasari†, Renze Ma , Ivan Hung‡, Zhehong Gan‡, Jeffery W. Kelly§, Michael C. Fitzgerald

For Table of Contents Use Only


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Pathogenic Mutations Induce Partial Structural Changes in the Native

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