Intra- and Intersubunit Ion-Pair Interactions Determine the Ability of

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Intra- and inter-subunit ion-pair interactions determine the ability of apolipoprotein C-ll mutants to form hybrid amyloid fibrils Nevena Todorova, Courtney O. Zlatic, Yu Mao, Irene Yarovsky, Geoffrey John Howlett, Paul R. Gooley, and Michael D. W. Griffin Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01146 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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Biochemistry

Intra- and inter-subunit ion-pair interactions determine the ability of apolipoprotein C-ll mutants to form hybrid amyloid fibrils Nevena Todorova1§, Courtney O. Zlatic2§, Yu Mao2§, Irene Yarovsky1, Geoffrey J. Howlett2, Paul R. Gooley2, and Michael D. W. Griffin2*

1

School of Engineering, RMIT University, GPO Box 2476V, Melbourne, Victoria 3001,

Australia 2

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville,

Victoria 3010, Australia and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. §

These authors contributed equally to the work

*To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia. Tel.: 61-3-9035-4233; Fax: 61-3-93481421; e-mail: [email protected]

Running title: Ion-pair interactions in amyloid fibrils

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Abbreviations: apo, apolipoprotein; WT, wild-type; MD, molecular dynamics; ThT, thioflavin T; GuHCl, guanidine hydrochloride; KDDK, K30D-D69K; AAU, acetic acid-urea.

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Abstract The apolipoprotein family is structurally defined by amphipathic α-helical regions that interact with lipid surfaces. In the absence of lipid, human apolipoprotein (apo) C-II also forms welldefined amyloid fibrils with cross-β structure. Formation of this β-structure is accompanied by the burial of two charged residues, K30 and D69 that form an ion-pair within the amyloid fibril core. Molecular dynamics (MD) simulations indicate these buried residues form both intra- and inter-subunit ion-pair interactions that stabilize the fibril. Mutations of the ion-pair (either K30D or D69K) reduce fibril stability and prevent fibril formation by K30D apoC-II under standard conditions. We investigated whether mixing K30D apoC-II with other mutants would overcome this loss of fibril forming ability. Co-incubation of equimolar mixtures of K30D apoC-II with wild-type, D69K or a double mutant (K30D, D69K) apoC-II promoted the incorporation of K30D apoC-II into hybrid fibrils with increased stability. MD simulations showed an increase in inter-subunit ion-pair interactions accompanied the increased stability of the hybrid fibrils. These results demonstrate the important role of both intra- and inter-subunit charge interactions in stabilizing apoC-II amyloid fibrils, a process that may be a key factor in determining the general ability of proteins to form amyloid fibrils.

Keywords: intermolecular interactions, amyloid fibrils, molecular dynamics, apolipoprotein C-II, cross-beta structure, heterologous, hybrid, ion-pair, charge-pair, salt bridge, protein misfolding

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Amyloid deposits accumulate in a range of human diseases including Alzheimer’s and Parkinson’s disease and type II diabetes 1. These deposits are characterized by the presence of protein fibrils, called amyloid fibrils, as well as several non-fibrillar components including lipids, proteoglycans and serum amyloid P 2, 3. The formation of amyloid fibrils occurs when specific proteins misfold and spontaneously aggregate 4. These fibrils exhibit fluorescence in the presence of thioflavin T, bind Congo Red and have a cross-β structure identified by X-ray diffraction 1. Prevalent among the proteins known to form amyloid fibrils in vivo are several members of the apolipoprotein family including apolipoprotein (apo) A-I, apoA-II, apoC-II, apoC-III, apoE, serum amyloid A and the apolipoprotein-like α-synuclein 5, 6. This conserved family of proteins is characterized by the presence of class A amphipathic helical regions that mediate lipid binding 7. An age-related increase in localized amyloid deposits within the artery wall 8 as well as the immunohistochemical localization of several apolipoproteins within aortic atherosclerotic lesions suggests a link with atherosclerosis progression 9. It has been proposed that the widespread occurrence of apolipoproteins within amyloid plaques is due to their limited conformational stability in the lipid-free state leading to misfolding and aggregation to form insoluble amyloid fibrils 10. Human apoC-II serves as a relevant model to study the formation of amyloid fibrils by apolipoproteins. ApoC-II is found co-localized to the amyloid marker, serum amyloid P, in atherosclerotic plaques and has recently been implicated in a novel type of renal amyloidosis 11, 12

. ApoC-II in vitro readily self-associates into amyloid fibrils with individual subunits adopting

a ‘letter G-like’ β-strand-loop-β-strand conformation 13. All-atom molecular dynamics (MD) simulations in explicit solvent showed that the model has a stable cross-β core with a flexible connecting loop devoid of persistent secondary structure. This model suggested that the fibrils 4


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formed by apoC-II are composed of one molecule per 4.7 Å rise of the cross-β structure, packed with parallel β-strands in-register. A key structural feature of the model is the presence of a buried ion-pair between K30 and D69 within the fibril core. The formation of a buried K30-D69 ion-pair within apoC-II amyloid fibrils reconciles the dual abilities to form amphipathic helices or cross-β structure in the presence or absence of lipid, respectively 5. An intermolecular salt bridge has recently been reported for the amyloid core region of α-synuclein fibrils 14 while modelling studies have suggested an inter-subunit ion-pair located within the core of apoA-I 15. The capacity to form a buried ion-pair may be a general property of those members of the apolipoprotein family that form amyloid fibrils. Mutations of the K30-D69 ion-pair in apoC-II confirm the importance of this ion-pair in fibril formation. The mutations K30D and D69K reduce fibril stability and disrupt β-strand formation in the outer β-sheet around position 30 5, 16. Paradoxically, D69K apoC-II and the double mutant K30D, D69K (KDDK) apoC-II form fibrils more rapidly than wild-type (WT) apoC-II while K30D apoC-II does not form fibrils under standard fibril forming conditions (0.3 mg/ml apoC-II, 100 mM sodium phosphate buffer pH 7.4, 0.1% azide, 22 °C). We have attributed this finding to an inhibitory effect of D69 on fibril formation that is released when this residue is mutated to K69 17. Consideration of the intra- and inter-subunit interactions between positions 30 and 69 in apoC-II fibrils shows that homogeneous fibrils composed solely of the K30D apoC-II mutant have repulsive intra- and inter-subunit interactions between aspartate residues located at these positions. On the other hand, heterologous or hybrid fibrils composed of alternating K30D and WT apoC-II subunits would potentially include two attractive inter-subunit interactions and one attractive intra-subunit interaction. Similarly, hybrid fibrils with alternating KDDK and K30D apoC-II subunits would also include two attractive inter-subunit interactions and one attractive 5


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intra-subunit interaction. In contrast, hybrid fibrils with alternating K30D and D69K apoC-II subunits would allow four attractive inter-unit interactions but no attractive intra-subunit interactions. In the present study, we have investigated the relative importance of inter- and intra-subunit ion-pair interactions within amyloid fibrils by determining the relative abilities of the fibril inactive K30D mutant to be incorporated into fibrils when mixed in equal proportions with WT, KDDK or D69K apoC-II. MATERIALS AND METHODS Expression, mutagenesis and purification of apoC-II cDNA for D69K, K30D and KDDK apoC-II were constructed using procedures described previously 18. Mutations were verified by DNA sequencing (Applied Diagnostics, Victoria, Australia). WT and mutant apoC-II expression and purification was performed as described previously 19. All WT and mutant apoC-II preparations were analyzed using a QTOF LC mass spectrometer (Agilent Technologies Inc., DE) to verify correct molecular masses. Purified apoCII preparations were stored at -20 °C in 5 M guanidine hydrochloride (GuHCl) and 10 mM Tris.HCl, pH 8.0 as 30 - 40 mg/mL stocks Fibril formation Fibril formation was initiated by dilution of protein stocks (30 - 40 mg/mL) to 0.3 or 0.6 mg/mL in refolding buffer (100 mM sodium phosphate buffer pH 7.4, 0.1% azide) followed by incubation at 30 °C. Residual GuHCl present under folding conditions had little effect on the kinetics of fibril formation 20. Fibril formation was monitored using a continuous thioflavin T (ThT) assay with a final volume of 200 µL and 10 µM ThT 21 in a Paradigm Plate Reader (Beckman Coulter, USA) with 444 nm excitation and 485 nm emission filters, or a FLUOstar 6


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OPTIMA Plate Reader (BMG LABTECH, Germany) with 440-10 nm excitation and 480-10 nm emission filters. A centrifugal procedure was also used to monitor apoC-II fibril formation. Samples (180 µL) were centrifuged at 100,000 rpm for 30 minutes using a TLA-100 rotor in an OptimaMax centrifuge (Beckman Coulter, USA) and the supernatant and pellet fractions collected. The pellet fraction was resuspended in an equal volume of 5 M GuHCl, pH 8 for four hours to allow the fibrils to dissociate into monomers. Optical absorbance measurements at 280 nm were used to determine the amounts of protein in the pellet and supernatant fractions. Transmission Electron Microscopy Fibrils were formed at 30 °C by incubating equimolar concentrations (0.3 mg/mL) of K30D with WT, KDDK and D69K apoC-II in 100 mM sodium phosphate buffer, pH 7.4, 0.1% sodium azide. Carbon-coated copper grids were glow discharged for 15 seconds prior to sample application. The fibrils were diluted with milliQ water to 0.1 mg/mL, applied to grids, and allowed to adsorb for 1 minute. Samples were then blotted from the grid, stained twice with 2% potassium phosphotungstate, pH 6.8, and air-dried. Grids were examined at the Bio21 Electron Microscopy Unit using a FEI Tecnai G2 TF20 transmission electron microscope (FEI-Company, Eindhoven, The Netherlands), and a Gatan US1000 2k×2k CCD Camera (Pleasanton, CA, USA) was used to aquire digital images. Acetic acid-urea electrophoresis Acetic acid-urea (AAU) gel electrophoresis was used to separate apoC-II derivatives based on their net charge at pH 3. The AAU gel contained 20% bis-acrylamide solution (67:1), 2.5 M urea and 0.9 M acetic acid and was pre-electrophoresed at 160 V for two hours until the current fell to a steady level. Running buffer contained 0.9 M glacial acetic acid. Aliquot, pellet, or supernatant fractions of 20 µL were mixed with an equal volume of denaturing buffer (0.2% pyronin Y, 5 M 7


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urea, 1 M HCl) over 2 h and 15 µL was loaded onto the gel. The gel was run at 50 V for 10 h, stained with 0.25% Coomassie blue and then destained overnight with a solution of 10% methanol and 0.9 M acetic acid. Liquid chromatography mass spectrometry ApoC-II samples (10 µL of 0.3 mg/mL) were desalted by injecting into a Phenomenex C8 column (4 x 3 mm) connected to an Agilent 1100 LC TOF mass spectrometer (Santa Clara, CA, USA). Protein was eluted over a gradient of 0 - 60% acetonitrile, 0.1% formic acid for 20 min. MS spectra were analyzed using the in-built Agilent Protein software. Analysis of fibril stability GuHCl denaturation assays were used to analyze the relative stability of fibrils essentially as described previously22. Fibrils were formed at 37°C over 7 days at 0.9 mg/mL in order to reduce the time required for fibril growth. Samples were then diluted to 0.3 mg/mL with GuHCl at specific concentrations, vortexed, and kept for 24 h at room temperature before centrifugation at 100,000 rpm for 30 minutes using a TLA-100 rotor in an OptimaMax centrifuge (Beckman Coulter, USA). Directly following centrifugation, the supernatants were removed and optical absorbance measurements at 280 nm were recorded to determine protein concentration using a DU-800 UV/Vis Spectrophotometer (Beckman Coulter, USA). The amount of apoC-II in each supernatant sample was determined relative to uncentrifuged apoC-II in 6 M GuHCl. Fits to a 4parameter sigmoid function were used to derive the GuHCl concentrations at half-maximum dissociation (c1/2) for both WT and K30D apoC-II. Molecular dynamics simulations

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Hybrid apoC-II fibril tetramers composed of alternating subunits of K30D apoC-II with either WT, D69K or KDDK subunits, referred to as WT+K30D, D69K+K30D and KDDK+K30D tetramers, were subjected to molecular dynamics (MD) simulations in explicit solvent. Simulations were also performed on homogeneous KDDK tetramers. The starting structure used for the all-atom simulations was the stable full-length WT apoC-II fibril tetramer (Supporting Information, Figure S1A) described in our previous work 13. The K30D apoC-II model was made by substituting the lysine (K) at position 30 with an aspartate (D). The D69K apoC-II model was built by replacing D at position 69 with a K, while double K30D and D69K substitutions were made to construct the KDDK apoC-II model. All titratable residues were modeled as charged, representative of neutral conditions. Two different subunit arrangements, ABAB and BABA, where A and B represent different subunits, were considered for each hybrid tetramer to enhance the conformational sampling of each fibril (Figure S1B). The GROMACS 4.6.5 (www.gromacs.org) software suite 23 was employed for all MD simulations, while the simulation parameters and system set-up was similar to previous work 5, 16. The GROMOS force field parameter set 43A1 24 and the SPC water model 25 were used to model the protein and water pair-wise interactions. In all simulations, period boundary conditions were applied with switch cutoffs to calculate the Coulomb, and van der Waals interactions at 10 Å. The long-range electrostatic interactions were evaluated with the particle-nesh Ewald (PME) method. Periodic image (cross-cell) interactions were avoided by enclosing each apoC-II tetramer model in a sufficiently large cubic box with a minimum distance between the solute and the edges of the box of 12 Å. Approximately 72,000 SPC water molecules, corresponding to water density of ~1.0 g/cm3, were used to solvate the simulation box. A neutral simulations cell was ensured by adding counter ions to counteract any charge present in the system. The MD 9


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simulations were conducted under constant particle number, pressure and temperature (NPT) ensemble. The velocity rescaling method 26 was employed to maintain constant 300 K temperature of the system, with a temperature coupling constant of 0.1 ps. Constant pressure of 1 bar was performed using the Parrinello-Rahman scheme 27 and pressure coupling constant of 1.0 ps. The LINCS algorithm 28 was employed to constrain all bonds to their equilibrium length, which allowed a 2 fs integration time-step. To discard any steric clashes each system underwent energy minimization using the steepest descent approach. A 100 ps of MD simulation was performed with the protein fibril restrained to allow for the solvent to relax around the protein. Following the short equilibration, unrestrained simulation was performed for 300-350 ns for each system, and a total of 1.8 µs of data was collected. The last 50 ns of simulation were used for data analysis of each system, after the total energy and RMSD has plateaued. The visual molecular dynamics (VMD) software 29 was employed for the visualization of system geometries and interactions, and analysis of the protein secondary structure and ion-pair formation. The secondary structure of the middle two subunits from each tetramer was determined using the STRIDE algorithm and the data was averaged from the two subunit arrangements simulated to take into account any conformational variations. The criterion for the formation of an ion-pair was that the distance between any oxygen atoms of an acidic residue side-chain and the nitrogen atoms of a basic residue side-chain was within 3.2 Å. Cluster analysis was also performed to determine the most favorable equilibrium structure using the GROMOS clustering method and a backbone cutoff of 2.0 Å.

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RESULTS Incubation of K30D apoC-II monomer with WT, D69K or KDDK monomer The potential for K30D apoC-II to incorporate into fibrils with different variants of apoC-II was explored by mixing equimolar amounts of K30D apoC-II with WT, D69K and KDDK apoC-II under standard fibril forming conditions (Figure 1). K30D apoC-II alone at 0.6 mg/mL showed no change in ThT fluorescence, confirming its inability to form fibrils under the conditions used 17

, while WT, D69K and KDDK apoC-II all formed fibrils at both 0.3 mg/mL and 0.6 mg/mL.

The equimolar mixtures of K30D apoC-II with WT, D69K or KDDK all showed a timedependent increase in ThT fluorescence suggesting successful fibril formation. The maximum change in ThT fluorescence of these mixtures was higher than that of fibrils formed by WT, D69K or KDDK alone at 0.3 mg/mL, suggesting the incorporation of K30D into WT, D69K or KDDK apoC-II hybrid fibrils.

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Figure 1. ThT fluorescence analysis of apoC-II fibrils incubated under standard fibril forming conditions (100 mM sodium phosphate buffer pH 7.4 at 30 °C). The data for K30D apoC-II alone at 0.6 mg/mL are presented as red triangles. WT (A), D69K (B) and KDDK (C) apoC-II alone at 0.3 mg/mL are presented as open circles and at 0.6 mg/mL as filled circles, while mixtures of K30D apoC-II at 0.3 mg/mL with equimolar WT, D69K or KDDK apoC-II at 0.3 mg/mL, for a final concentration of 0.6 mg/mL, are shown as open orange diamonds. Data are the mean of two separate measurements. Error bars represent the standard deviation of the mean. 12


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To determine whether K30D apoC-II was incorporated into these fibrils, a pelleting assay was performed, where fibrils were pelleted at high speeds and separated from soluble protein. The proportions of total protein found in the pellet fractions of WT, D69K and KDDK apoC-II alone at 0.3 mg/mL were 86, 95 and 77%, respectively. The proportions of total protein found in the pellet fractions for fibrils formed by mixing WT, D69K and KDDK apoC-II at 0.3 mg/mL with equimolar concentrations of K30D apoC-II were 78, 79 and 72%, respectively. These results show that the pellet fractions of WT+K30D, D69K+K30D and KDDK+K30D apoC-II mixtures all comprise over 50% of the total protein in each case, demonstrating that K30D apoC-II was incorporated into the fibrils. The supernatant fraction for K30D apoC-II alone at 0.6 mg/mL contained approximately 100% of the total protein again confirming that apoC-II K30D does not form fibrils under these conditions. Transmission electron microscopy was also used to assess all samples to confirm the morphology of the aggregates observed during ThT analysis (Figure 2). The WT+K30D, D69K+K30D and KDDK+K30D apoC-II mixtures all showed a twisted ribbon fibril morphology, characteristic of homogeneous WT, D69K, KDDK and K30D apoC-II fibrils 19

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Figure 2. Electron micrographs of hybrid fibrils formed by equimolar mixtures of WT+K30D (A), KDDK+K30D (B), and D69K+K30D (C) apoC-II variants. Fibrils were grown at 0.6 mg/mL and incubated at 30 °C. Scale bar represents 50 nm. 13


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Analysis of hybrid fibrils using acetic acid-urea gel electrophoresis To further characterize the incorporation of K30D apoC-II into the observed hybrid fibrils, AAU gel electrophoresis analysis was conducted following the pelleting assay (Figure 3). WT and KDDK apoC-II were predicted to have similar mobility, while the bands for the D69K and K30D variants were predicted to migrate faster and slower, respectively, consistent with their predicted isoelectric points of 4.48 (WT), 4.96 (D69K), 4.58 (KDDK) and 4.31 (K30D) calculated using the program ProtParam 30. WT, D69K, and KDDK apoC-II incubated at concentrations of 0.3 mg/mL show little protein in the supernatant fraction, and a distinct band in the pellet fraction, indicating that the majority of protein was incorporated into fibrils. Furthermore, D69K apoC-II migrated faster as expected, validating this analysis for the isolation and identification of the apoC-II variants. ApoC-II variant K30D incubated at 0.3 mg/mL shows a strong band in the supernatant fraction and little protein in the pellet fraction, confirming that K30D apoC-II does not form fibrils and remains in the soluble fraction under these conditions. In contrast, samples of 0.3 mg/mL K30D apoC-II incubated in the presence of 0.3 mg/mL WT, D69K and KDDK apoC-II show significant bands corresponding to K30D apoC-II in the pellet fraction, indicating that this variant is incorporated into hybrid fibrils. Incubation of WT+K30D, D69K+K30D, and KDDK+K30D apoC-II resulted in a distinct band in the pellet fraction corresponding to K30D apoC-II, and a weak K30D apoC-II band in the supernatant fraction. This indicates that, while the majority of K30D apoC-II is incorporated into the pelleted fibrils, a significant proportion remains in the soluble fraction. In the case of the WT+K30D apoC-II mixture the pellet band for K30D appears more intense, and the supernatant band less intense, than for the D69K+K30D and KDDK+K30D apoC-II mixtures, suggesting that a higher proportion of K30D is incorporated into fibrils formed with WT apoC-II. A control sample 14


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showed that apoC-II K30D incubated at 0.6 mg/mL remained in the supernatant fraction and does not form fibrils at this higher concentration. These results confirm the ability of K30D apoC-II to incorporate into hybrid fibrils.

Figure 3. Acetic acid-urea PAGE analysis of hybrid fibrils formed by the equimolar mixtures of WT+K30D, D69K+K30D, and KDDK+K30D apoC-II variants. Samples of WT, D69K, K30D and KDDK apoC-II alone at 0.3 mg/mL (lanes 1-4) and samples at a total concentration of 0.6 mg/mL of equimolar mixtures of WT+K30D, D69K+K30D, K30D+K30D, and KDDK+K30D apoC-II (lanes 5-8) were incubated under fibril forming conditions (100 mM sodium phosphate buffer pH 7.4 at 30 °C for 30 h). The samples were centrifuged at 100,000 rpm (436,000×g) for 30 min and the supernatant fractions (top panel) and pellet fractions (bottom panel) for each sample were loaded onto an acetic acid-urea PAGE gel for analysis.

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Analysis of hybrid fibrils using mass spectrometry The pellet fractions of the hybrid fibrils were further analyzed by mass spectrometry to detect the presence of each apoC-II variant by their corresponding mass (Figure 4). Results for WT+K30D hybrid fibrils (Figure 4A) showed peaks at 8915 Da, corresponding to WT, and 8902 Da, corresponding to K30D apoC-II. Similar analyses of D69K+K30D (Figure 4B) and KDDK+K30D (Figure 4C) pellet fractions showed peaks corresponding to K30D apoC-II with D69K (8938 Da) and KDDK apoC-II (8915 Da), respectively. The peak corresponding to K30D apoC-II in the WT+K30D fibrils appeared larger than for D69K+K30D and KDDK+K30D apoC-II mixtures, broadly consistent with the band staining intensities in Figure 3 suggesting that K30D apoC-II incorporation into fibrils with WT apoC-II is more efficient. While different proteins are ionized to varying extents and the subsequent detection of the charged particles by mass/charge ratio is varied, mass spectrometry performed in this way can be used as a qualitative measurement of relative protein amounts present where detection of the apoC-II variants is assumed similar. Additional peaks for each protein derivative are observed at approximately +131 Da (WT and KDDK, 9046 Da; K30D, 9033 Da; D69K, 9057 Da) due to incomplete cleavage of the N-terminal methionine in the expression host. The presence of this methionine residue has not been found to interfere with fibril formation studies. These results further confirm findings that K30D apoC-II successfully incorporates into hybrid fibrils with each variant.

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Figure 4. Mass spectra for the pellet fractions of hybrid fibrils. Equimolar mixtures of WT (A), D69K (B), or KDDK (C) with K30D at a total concentration of 0.6 mg/mL were incubated in 100 mM sodium phosphate buffer pH 7.4 at 30 °C for 30 h. The samples were then centrifuged at 100,000 rpm (436,000×g) to separate the fibrils. Pellet fractions were analyzed individually by mass spectrometry. The deconvoluted mass spectrum of the pellet is shown with a normalized intensity.

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Incubation of K30D apoC-II monomer with preformed fibrils The ability of monomeric K30D apoC-II to incorporate into preformed fibrils was investigated by mixing preformed WT, D69K or KDDK apoC-II fibrils (0.3 mg/mL) with different apoC-II variant monomers at equimolar concentration. Preformed WT, D69K or KDDK apoC-II fibrils alone at 0.3 mg/mL exhibited no changes in ThT fluorescence over the incubation time of the assay, indicating that preformed fibrils were stable under these conditions (Figure 5). ApoC-II monomers of each variant were added to preformed fibrils of the same variant at equimolar concentrations, to create homogeneous fibrils. The results for each sample showed a timedependent increase in ThT fluorescence indicating incorporation of monomer into the preformed fibrils. To assess if apoC-II K30D was able to incorporate into preformed fibrils to yield hybrid fibrils, apoC-II K30D monomer was also added to preformed fibril samples of each variant at equimolar concentrations. A time-dependent increase in ThT fluorescence for these mixed fibrils was also observed, indicating incorporation of monomeric K30D apoC-II into preformed fibrils. However, the samples of preformed fibrils with added identical variant had a greater rate of change in ThT fluorescence compared to samples of preformed fibril with added K30D apoC-II variant. This indicates that monomeric K30D apoC-II is incorporated into preformed fibrils more slowly than when variant apoC-II monomers are used to extend preformed fibrils of the same variant.

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Figure 5. The incubation of K30D monomers with preformed apoC-II fibrils. Preformed fibrils of WT (A), D69K (B) or KDDK (C) alone at 0.3 mg/mL are shown as open circles and preformed fibrils mixed with apoC-II monomers of the same variant to a final concentration of 0.6 mg/mL are shown as filled circles. Preformed fibrils mixed with K30D monomers to a final concentration of 0.6 mg/mL are shown as open orange diamonds. Data are the mean of two separate measurements. Error bars represent the standard deviation of the mean.

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Analysis of fibril stability The relative stabilities of the hybrid apoC-II fibrils were determined using GuHCl denaturation assays 22. As previously reported, it is possible for K30D apoC-II to form fibrils at high concentrations 16. Fibril samples of WT, K30D and D69K apoC-II alone and WT+K30D and D69K+K30D apoC-II hybrid fibrils were prepared by incubating samples for 4 days at 37 °C at 0.9 mg/mL. The results in Figure 6A show distinct denaturation profiles for apoC-II WT and K30D fibrils alone, as previously reported 16. K30D apoC-II fibrils exhibit reduced stability in comparison to WT apoC-II fibrils. The results for hybrid WT+K30D fibrils show an equilibrium unfolding curve intermediate between the curves for the WT and K30D apoC-II fibrils alone. Similarly, the results in Figure 6B show K30D fibrils have reduced stability compared to D69K fibrils. The results for the hybrid D69K+K30D fibrils indicate the equilibrium unfolding curve for these hybrid fibrils lies between the curves obtained for K30D and D69K fibrils alone. These results indicate that the formation of K30D apoC-II hybrid fibrils with either WT or D69K apoCII leads to an increase in fibril stability when compared to K30D fibrils alone and reduced stability when compared to WT or D69K apoC-II fibrils alone.

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Figure 6. Analysis of fibril stabilities. Preformed apoC-II fibrils K30D alone (filled circles), WT alone (open circles) and WT+K30D hybrid fibrils (grey circles) (Panel A) and K30D alone (filled circles), D69K alone (open circles) and D69K+K30D hybrid fibrils (grey circles) (Panel B), were incubated in the presence of various concentrations of GuHCl for 24 h. ApoC-II present in the supernatants was estimated by optical absorbance measurements at 280 nm following centrifugation at 100,000 rpm (436,000×g) for 30 min. Data are shown as fractions relative to uncentrifuged samples in 6 M GuHCl. Solid lines represent best-fits to the data using a 4parameter sigmoid function.

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Molecular dynamics analysis of K30D apoC-II hybrid fibrils We performed MD simulations to study the structural changes associated with the formation of K30D apoC-II hybrid fibrils. Visual inspection of the simulated trajectories and snapshots of the equilibrium conformations showed all hybrid fibrils retained the inherent “letter-G-like” fibril structure (Figure 7). Quantitative characterization of cross-β structure within the hybrid tetramers was obtained by determining the fraction of time each residue spent in the β-strand conformation at equilibrium (Figure 8). The results for WT+K30D tetramers and D69K+K30D tetramers showed similar and stable β-sheet localization, where the outer β-sheet (β-sheet 1) spanned between residues 22 and 34, and the inner β-sheet (β-sheet 2) between residues 61 and 75. In contrast, the data for KDDK+K30D tetramers showed retention of β-structure in the inner β-sheet and between residues 22 and 28 of the outer β-sheet but no persistent β-structure around position 30 suggesting considerable structural perturbation within this region. The composition of the charged residues at position 30 and 69 is the same in the WT+K30D and KDDK+K30D tetramers. This suggests that the presence of negative charges at position 30 in the outer β-sheet of the KDDK+K30D tetramers destabilizes the outer β-sheet structure more than when the anionic residues are located at position 69 within the inner sheet of WT+K30D tetramers.

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Figure 7. Molecular dynamics snapshots of the equilibrium conformation from the WT+K30D (A), D69K+K30D (B) and KDDK+K30D (C) and KDDK (D) tetramer simulations. The most favorable cluster population is also shown in parentheses. The typical structure and arrangement of the titratable residues at positions 30 and 69 are shown as insets. The residues are represented as liquorice and colored blue (Lys) and red (Asp), respectively. The strands and respective residues shown in parenthesis are also labeled for each tetramer. 23


Biochemistry

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Figure 8. Secondary structure (β-strand) content determined from the molecular dynamics simulations of the hybrid apoC-II fibril tetramers. The average percentage time each residue spends in the β-strand conformation at equilibrium for the WT+K30D (A), D69K+K30D (B), KDDK+K30D (C) and KDDK+KDDK (D) tetramer simulations.

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Detailed examination of the structures around positions 30 and 69 showed significant differences between the various hybrid structures. As expected, the WT+K30D tetramers exhibited intrasubunit electrostatic attraction between WT residues (D69-K30) and inter-subunit attractions between the neighboring (K30-D30 and K30-D69) residues (where K30D residues are in bold type, Table 1). The inter-strand interactions between the bulky K30 and D69 shielded the likecharge repulsions between the aspartates at position 69 (Figure 7) to prevent loss of β-structure. The D69K+K30D apoC-II tetramers exhibited only inter-strand ion-pair interactions (Table 1). The electrostatic attraction between neighboring residues in the outer β-sheet (K30-D30) and inner β-sheet, (K69-D69) were more transient in nature with 5-30% occurrence (Table 1) compared to the more persistent competing ion-pair interactions between residues in opposing βsheets (D69-K30 and D30-K69). The simulated structures showed the outer β-sheet shifted laterally by ~4.8 Å with respect to the inner β-sheet, to accommodate these electrostatic attractions. Ion-pair analysis for the KDDK+K30D tetramers showed no intra-subunit electrostatic interactions and one persistent inter-subunit interaction between K69 and D69 residues of neighboring strands (Table 1). This observation is consistent with the loss of βstructure in the outer β-sheet reported above (Figure 8). To examine this further, a control simulation was performed for homogeneous KDDK tetramers. The results showed only minor loss in β-stand structure around position 30 (Figure 8) and retention of an intra-subunit ion-pair interaction between K30 and D69 in the KDDK subunit (Table 1). This suggests that the anionic D69 from the K30D subunit destabilizes the outer β-sheet of KDDK+K30D hybrid tetramers. The observation that homogenous KDDK tetramers have reduced β-strand content near D30, compared to homogeneous WT tetramers 13, suggests the presence of an anionic residue at position 30 further destabilizes the fibril structure.

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Table 1: Electrostatic interactions between titratable residues at positions 30 and 69 of hybrid fibrils formed between K30D and other apoC-II variants Molecular Interaction Typeb

Electrostatic Interactionsa 5-30%c

> 30%c

WT+K30D apoC-II fibrils Intra

D69–K30

n.d.d

Inter

D30-K30

D69–K30

D69K+K30D apoC-II fibrils Intra

n.d.

n.d.

Inter

D30-K30 D69-K69

D69-K30 D30-K69

KDDK+K30D apoC-II fibrils Intra

n.d.

n.d.

Inter

n.d.

D69-K69

Homogeneous KDDK apoC-II fibrils Intra

D30-K69

n.d.

Inter

n.d

D30-K69

a

MD simulations were performed on hybrid apoC-II fibril tetramers composed of alternating subunits of K30D apoC-II with either WT, D69K or KDDK subunits. Control data for homogeneous KDDK apoC-II fibrils have also been included. An ion-pair was considered formed if the distance between any oxygen atoms of an acidic residue side-chain and the nitrogen atoms of a basic residue side-chain was within a distance of 3.2 Å. Residues belonging to the K30D strand are in bold type. b

Intra- and inter-subunit electrostatic interactions were analyzed for the middle-two strands of each fibril tetramer. c

The persistence of electrostatic interactions (5-30% and >30%) were calculated for the last 50 ns of the simulations.

d

n.d.: none detected 26


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DISCUSSION The inability of K30D apoC-II to form amyloid fibrils under standard fibril forming conditions is consistent with MD simulation results that show intra- and inter-subunit electrostatic repulsion between aspartate residues located at positions 30 and 69 16. This finding afforded an opportunity to examine the effect of adding potential ion-pair partners at these positions by mixing K30D apoC-II with other apoC-II mutants and testing for the formation of hybrid fibrils. The results showed extensive incorporation of K30D apoC-II (0.3 mg/ml) into hybrid fibrils when mixed with equal concentrations (0.3 mg/ml) of WT, D69K or KDDK apoC-II (Figure 1). The results of preparative ultracentrifuge, gel electrophoresis and mass spectrometry analysis confirmed that the majority of K30D apoC-II in the mixtures is incorporated into hybrid fibrils (Figures 3 and 4). Analysis of the stability of the hybrid fibrils WT+K30D and D69K+K30D apoC-II showed a significant increase in stability compared with homogeneous K30D fibrils formed at higher concentrations. These results indicate a direct effect of adding potential ionpair partners to K30D apoC-II on both the rate of fibril formation and the stability of the fibrils formed. MD simulations of WT tetramers at neutral pH identified both intra- and inter-subunit ion-pair formation between K30 and D69 16. Similarly, simulations of KDDK tetramers demonstrate both intra- and inter-subunit ion-pair interactions occur between the oppositely charged residues D30 and K69 (Table 1). Loss of these ion-pair interactions in K30D and D69K apoC-II tetramers would account for the observed reduction in stabilities of fibrils formed from these mutants. MD simulations were used to determine the extent of intra- and inter-subunit interactions within the hybrid fibrils. For WT, D69K and KDDK hybrid tetramers with K30D apoC-II there was an increase in the number of inter-subunit ion-pair interactions of 2, 4 and 1 27


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respectively (Table 1). In considering the persistence of electrostatic interactions, it should be noted that many of the interactions observed for specific hybrids are mutually exclusive. For instance, in the case of WT+K30D hybrids, K30 of WT can form an intra-subunit ion-pair interaction with D69 or inter-subunit interactions with D30 or D69 of the K30D subunit. In general, inter-subunit ion-pair interactions between the inner and outer β-sheets were more persistent than ion-pair interactions within the same β-sheet. The calculated persistence frequencies in Table 1 were limited to interactions between the middle two subunits of the tetramer and do not take into consideration competing interactions with the flanking subunits of the tetramer. The persistence frequencies of the inter-subunit ion-pair interactions are therefore an under-estimate of the overall extent of these interactions. The model chosen to simulate hybrid fibrils assumes alternating mutant subunits. Since WT and mutant apoC-II form fibrils at different rates it is likely that at least in the early stages homogeneous fibrils composed of the rapidly forming mutant will exist which then seed the incorporation of the slower forming mutant into mixed fibrils. In the latter stages of fibril formation, it is possible that existing fibrils will facilitate the formation of regions composed solely of the slower forming mutant. Over time however, apoC-II fibrils undergo extensive breaking and joining to approach an equilibrium distribution of fibrils 31. Under these conditions, it seems likely that sections of the fibrils will contain a pattern of alternating mutant subunits and that this would represent a stable arrangement. In focusing on positions 30 and 69 of the hybrid tetramers it should be pointed out that other ion-pair and hydrogen bonding interactions have been identified in homogeneous WT and K30D fibrils 16. These additional interactions include both intra- and inter-subunit interactions and lie outside the core cross-β regions of apoC-II fibrils. As these interactions are located outside the cross-β core and are conserved in WT and all of the apoC-II mutants studied,

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Biochemistry

they are assumed not to play a direct role in the ability of K30D apoC-II to form hybrid fibrils with WT, D69K and KDDK apoC-II. An important consideration concerning homogeneous K30D apoC-II fibrils is that the replacement of the hydrophobic methylene chain of K with the single methylene group in D leads to a loss of hydrophobicity and greater solvent accessibility 16. This factor may play a role in the formation of K30D apoC-II hybrids. However, the MD simulations demonstrate a direct involvement of both intra- and inter-subunit interactions in hybrid fibril formation. For KDDK and D69K hybrids with K30D apoC-II the results show an increase in inter-subunit interactions but no intra-subunit interactions indicating a major driving force for fibril formation is the ability to form inter-subunit ion-pair interactions. Hybrid or heterologous fibril formation is implicated in a number of human diseases. Mutations in the transthyretin (TTR) gene correlate to disease progression and lead to the incorporation of both WT and mutant TTR into the amyloid deposits of heterozygotes 32. The success of liver transplants to treat cardiac amyloidosis in patients carrying TTR mutations have been limited by the ability of WT TTR to continue incorporating into amyloid fibrils containing mutant TTR 32, 33. Specific point mutations in α-synuclein increase the incidence of Parkinson’s disease. Individuals who are heterozygous with one of these point mutations and WT α-synuclein form amyloid deposits containing both WT and mutant α-synuclein 34. In vitro studies show mutant forms of α-synuclein seed fibril formation by WT α-synuclein 34, 35. Similarly human lysozyme variants accelerate fibril formation of WT lysozyme 36. These studies serve to illustrate the widespread incidence of hybrid amyloid fibrils. The present work on apoC-II fibrils demonstrate heterologous fibril formation is driven by both intra- and inter-subunit ion-pair interactions within the amyloid core region of the fibrils. Such interactions may be a key determinant of the general ability of proteins to form amyloid fibrils. 29


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Funding information

I.Y and N.T. acknowledge the support from the NHMRC Centre of Research Excellence for Electromagnetic Bioeffects Research (CRE1042464). M.D.W.G is the recipient of an Australian Research Council Future Fellowship (project number FT140100544). This research was undertaken with the assistance of resources from the National Computational Infrastructure (NCI). This research was also supported by a Victorian Life Sciences Computation Initiative (VLSCI) (grant number VR0028) on its Peak Computing Facility located at the University of Melbourne, an initiative of the Victorian Government, Australia.

Supporting information

Figure S1: The starting structure of the hybrid apoC-II fibrils used for the molecular dynamics simulations.

References 1.

Sipe, J. D., and Cohen, A. S. (2000) Review: history of the amyloid fibril, J Struct Biol 130, 88-98.

2.

Alexandrescu, A. T. (2005) Amyloid accomplices and enforcers, Protein science : a publication of the Protein Society 14, 1-12.

3.

Pepys, M. B. (2006) Amyloidosis, Annu Rev Med 57, 223-241.

4.

Dobson, C. M. (2003) Protein folding and misfolding, Nature 426, 884-890.

5.

Mao, Y., Zlatic, C. O., Griffin, M. D., Howlett, G. J., Todorova, N., Yarovsky, I., and Gooley, P. R. (2015) Hydrogen/Deuterium Exchange and Molecular Dynamics Analysis of Amyloid Fibrils Formed by a D69K Charge-Pair Mutant of Human Apolipoprotein CII, Biochemistry 54, 4805-4814.

6.

de Messieres, M., Huang, R. K., He, Y., and Lee, J. C. (2014) Amyloid triangles, squares, and loops of apolipoprotein C-III, Biochemistry 53, 3261-3263. 30


Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

7.

Segrest, J. P., Jones, M. K., De Loof, H., Brouillette, C. G., Venkatachalapathi, Y. V., and Anantharamaiah, G. M. (1992) The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function, J Lipid Res 33, 141-166.

8.

Mucchiano, G., Cornwell, G. G., 3rd, and Westermark, P. (1992) Senile aortic amyloid. Evidence for two distinct forms of localized deposits, Am J Pathol 140, 871-877.

9.

Teoh, C. L., Griffin, M. D., and Howlett, G. J. (2011) Apolipoproteins and amyloid fibril formation in atherosclerosis, Protein Cell 2, 116-127.

10.

Hatters, D. M., Minton, A. P., and Howlett, G. J. (2002) Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II, The Journal of biological chemistry 277, 7824-7830.

11.

Nasr, S. H., Dasari, S., Hasadsri, L., Theis, J. D., Vrana, J. A., Gertz, M. A., Muppa, P., Zimmermann, M. T., Grogg, K. L., Dispenzieri, A., Sethi, S., Highsmith, W. E., Jr., Merlini, G., Leung, N., and Kurtin, P. J. (2016) Novel Type of Renal Amyloidosis Derived from Apolipoprotein-CII, Journal of the American Society of Nephrology : JASN DOI: 10.1681/ASN.2015111228.

12.

Stewart, C. R., Haw, A., 3rd, Lopez, R., McDonald, T. O., Callaghan, J. M., McConville, M. J., Moore, K. J., Howlett, G. J., and O'Brien, K. D. (2007) Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications, J Lipid Res 48, 2162-2171.

13.

Teoh, C. L., Pham, C. L., Todorova, N., Hung, A., Lincoln, C. N., Lees, E., Lam, Y. H., Binger, K. J., Thomson, N. H., Radford, S. E., Smith, T. A., Muller, S. A., Engel, A., Griffin, M. D., Yarovsky, I., Gooley, P. R., and Howlett, G. J. (2011) A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations, J Mol Biol 405, 1246-1266.

14.

Tuttle, M. D., Comellas, G., Nieuwkoop, A. J., Covell, D. J., Berthold, D. A., Kloepper, K. D., Courtney, J. M., Kim, J. K., Barclay, A. M., Kendall, A., Wan, W., Stubbs, G., Schwieters, C. D., Lee, V. M. Y., George, J. M., and Rienstra, C. M. (2016) Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein, Nat Struct Mol Biol 23, 409-415.

31


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

15.

Page 32 of 35

Louros, N. N., Tsiolaki, P. L., Griffin, M. D. W., Howlett, G. J., Hamodrakas, S. J., and Iconomidou, V. A. (2015) Chameleon 'aggregation-prone' segments of apoA-I: A model of amyloid fibrils formed in apoA-I amyloidosis, Int J Biol Macromol 79, 711-718.

16.

Mao, Y., Todorova, N., Zlatic, C. O., Gooley, P. R., Griffin, M. D. W., Howlett, G. J., and Yarovsky, I. (2016) Solution Conditions Affect the Ability of the K30D Mutation To Prevent Amyloid Fibril Formation by Apolipoprotein C-II: Insights from Experiments and Theoretical Simulations, Biochemistry 55, 3815-3824.

17.

Mao, Y., Teoh, C. L., Yang, S., Zlatic, C. O., Rosenes, Z. K., Gooley, P. R., Howlett, G. J., and Griffin, M. D. (2015) Charge and charge-pair mutations alter the rate of assembly and structural properties of apolipoprotein C-II amyloid fibrils, Biochemistry 54, 14211428.

18.

Binger, K. J., Griffin, M. D., and Howlett, G. J. (2008) Methionine oxidation inhibits assembly and promotes disassembly of apolipoprotein C-II amyloid fibrils, Biochemistry 47, 10208-10217.

19.

Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., and Howlett, G. J. (2000) Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops, Biochemistry 39, 8276-8283.

20.

Binger, K. J., Pham, C. L., Wilson, L. M., Bailey, M. F., Lawrence, L. J., Schuck, P., and Howlett, G. J. (2008) Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining, J Mol Biol 376, 1116-1129.

21.

Ryan, T. M., Griffin, M. D., Teoh, C. L., Ooi, J., and Howlett, G. J. (2011) High-affinity amphipathic modulators of amyloid fibril nucleation and elongation, J Mol Biol 406, 416429.

22.

Baldwin, A. J., Knowles, T. P. J., Tartaglia, G. G., Fitzpatrick, A. W., Devlin, G. L., Shammas, S. L., Waudby, C. A., Mossuto, M. F., Meehan, S., Gras, S. L., Christodoulou, J., Anthony-Cahill, S. J., Barker, P. D., Vendruscolo, M., and Dobson, C. M. (2011) Metastability of Native Proteins and the Phenomenon of Amyloid Formation, J Am Chem Soc 133, 14160-14163.

23.

Hess, B., Kutzner, C., van der Spoel, D., and Lindahl, E. (2008) GROMACS 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation, J. Chem. Theory Comput. 4, 435-447. 32


Page 33 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

24.

van Gunsteren, W. F., Billeter, S. R., Eising, A. A., Hünenberger, P. H., Krüger, P., Mark, A. E., Scott, W. R. P., and Tironi, I. G. (1996) Biomolecular simulation: the GROMOS96 manual and user guide, BIOMOS & vdf Hochschulverlag AG an der ETH Zurich, Groningen & Zürich.

25.

Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., and Hermans, J. (1981) Interaction models for water in relation to protein hydration, In Intermolecular Forces (Pullman, B., Ed.), pp 331-342, Springer, Dordrecht, Netherlands.

26.

Bussi, G., Donadio, D., and Parrinello, M. (2007) Canonical sampling through velocity rescaling, J Chem Phys 126, 14101.

27.

Parrinello, M., and Rahman, A. (1981) Polymorphic Transitions in Single-Crystals - a New Molecular-Dynamics Method, J Appl Phys 52, 7182-7190.

28.

Hess, B., Bekker, H., Berendsen, H. J. C., and Fraaije, J. G. E. M. (1997) LINCS: A linear constraint solver for molecular simulations, J Comput Chem 18, 1463-1472.

29.

Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD: Visual molecular dynamics, J Mol Graph Model 14, 33-38.

30.

Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., and A.;, B. (2005) Protein Identification and Analysis Tools on the ExPASy Server, In The Proteomics Protocols Handbook (Walker, J. M. E., Ed.), Humana Press.

31.

Yang, S., Griffin, M. D. W., Binger, K. J., Schuck, P., and Howlett, G. J. (2012) An Equilibrium Model for Linear and Closed-Loop Amyloid Fibril Formation, Journal of Molecular Biology 421, 364-377.

32.

Ihse, E., Suhr, O. B., Hellman, U., and Westermark, P. (2011) Variation in amount of wild-type transthyretin in different fibril and tissue types in ATTR amyloidosis, Journal of molecular medicine 89, 171-180.

33.

Yazaki, M., Tokuda, T., Nakamura, A., Higashikata, T., Koyama, J., Higuchi, K., Harihara, Y., Baba, S., Kametani, F., and Ikeda, S. (2000) Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin, Biochemical and biophysical research communications 274, 702-706.

34.

Ono, K., Ikeda, T., Takasaki, J., and Yamada, M. (2011) Familial Parkinson disease mutations influence alpha-synuclein assembly, Neurobiology of disease 43, 715-724.

33


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

35.

Page 34 of 35

Sidhu, A., Segers-Nolten, I., and Subramaniam, V. (2016) Conformational Compatibility Is Essential for Heterologous Aggregation of alpha-Synuclein, ACS chemical neuroscience 7, 719-727.

36.

Morozova-Roche, L. A., Zurdo, J., Spencer, A., Noppe, W., Receveur, V., Archer, D. B., Joniau, M., and Dobson, C. M. (2000) Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants, J Struct Biol 130, 339-351.

34


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For Table of Contents Use Only Intra- and inter-subunit ion-pair interactions determine the ability of apolipoprotein C-ll mutants to form hybrid amyloid fibrils Nevena Todorova, Courtney O. Zlatic, Yu Mao, Irene Yarovsky, Geoffrey J. Howlett, Paul R. Gooley, and Michael D. W. Griffin.

228x116mm (144 x 144 DPI)


Intra- and Intersubunit Ion-Pair Interactions Determine the Ability of

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