Conformational ensemble of native α-synuclein in solution as determined by short-distance crosslinking constraint-guided discrete molecular dynamics simulations
α-Synuclein is involved in the pathogenesis of misfolding-related neurodegenerative diseases , in particular Parkinson’s disease 1 , 2 ., A misfolding event leads to the formation of oligomers which are believed to result in cell toxicity and which eventually lead to the death of neuronal cells 3 ., α-Synuclein is thought to interact with lipid vesicles in vivo 4 and the toxicity is thought to be mediated via membrane disruption by misfolded oligomers 5 ., Moreover , a prion-like spread of the pathology via the conversion of native α-synuclein molecules by toxic oligomers has been suggested 6 ., Native α-synuclein is considered to be an intrinsically disordered protein , although there is evidence that some globular structure exists in solution , which may serve as a basis for understanding the mis-folding and oligomerization pathways ., A number of biophysical methods , such as NMR , EPR , FRET , and SAXS—in combination with computational methods—have been applied to the study of intrinsically disordered proteins , including the structure of α-synuclein in solution 7–10 ., In all of these cases , even a limited amount of experimental structural data was helpful in the characterization of the conformational ensemble of α-synuclein in solution ., Recently , we developed a method for determination of protein structures , termed short-distance crosslinking constraint-guided discrete molecular dynamics simulations ( CL-DMD ) , where the folding process is influenced by short-distance experimental constraints which are incorporated into the DMD force field 11 ., Adding constraints to DMD simulations results in a reduction of the possible conformational space and allows the software to achieve protein folding on a practical time scale ., We have tested this approach on well-structured proteins including myoglobin and FKBP and have observed clear separation of low-energy clusters and a narrow distribution of structures within the clusters ., The conformational flexibility of intrinsically disordered proteins , such as α-synuclein , brings additional challenges to the computational process 12 ., In cases like this , proteins exist as a collection of inter-converting conformational states , and crosslinking data represents multiple conformations of a protein rather than a single structure ., In addition , recent research indicates that traditional force fields with their parametrization are not ideal for providing an accurate description of disordered proteins , and tend to produce more compact structures 13 ., Recently research has been focused on improving traditional state-of-the-art force fields and their ability to predict structures of disordered proteins without losing their accuracy for structured proteins 14 ., In this work we use a Medusa force field 15–17 that is utilized in DMD simulations is discretized to mimic continuous potentials ., DMD uses a united atom representation for the protein where all heavy atoms and polar hydrogens are explicitly accounted ., The solvation energy is described in terms of the discretized Lazaridis-Karplus implicit solvation model 18 and inter-atomic interactions , such van der Waals and electrostatics , are approximated by a series of multistep square-well potentials ., Other additional potentials , such as pair-wise distance constraints 19 , 20 and solvent accessibility information 21 , 22 can also be readily integrated ., During CL-DMD simulations there are no continuous forces that would drive the atoms to satisfy all constraints , rather generating conformational ensembles , which satisfy an optimal number of the constraints are generated ., This , to some degree , naturally resolves conflicting experimental constraints ., Thus , CL-DMD simulations are a viable computational platform for the structural analysis of intrinsically disordered proteins 23 in general , and α-synuclein in particular ., Here , we used the CL-DMD approach 11 to determine conformational ensembles of the α-synuclein protein in solution ., During this process , α-synuclein was crosslinked with a panel of short-range crosslinkers , crosslinked proteins were enzymatically digested , crosslinked residues were determined by LC-MS/MS analysis , and the resulting data on inter-residues distances were introduced into DMD force field as external constraints ., To experimentally validate the predicted structures , we analyzed α-synuclein using surface modification ( SM ) , circular dichroism ( CD ) , hydrogen-deuterium exchange ( HDX ) , and long-distance crosslinking ( LD-CL ) ., α-Synuclein was crosslinked with a panel of short-range reagents azido-benzoic acid succinimide ( ABAS-12C6/13C6 ) , succinimidyl 4 , 4-azipentanoate ( SDA ) , 24 triazidotriazirine ( TATA-12C3/13C3 ) , 25 and 1-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide ( EDC ) 26 ., ABAS and SDA are hetero-bifunctional amino group-reactive and photo-reactive reagents , TATA is a homo-bifunctional photo-reactive reagent , and EDC is a zero-length carboxyl-to-amino group crosslinker ., Crosslinked proteins were digested with proteinase K or trypsin proteolytic enzymes , and the digest was analyzed by LC-MS/MS to identify crosslinked peptides ( S1 Table ) ., We used an equimolar mixture of 14N- and 15N-metabolically labeled α-synuclein to exclude potential inter-protein crosslinks from the analysis and to facilitate the assignment of crosslinked residues based on the number of nitrogen atoms in the crosslinked peptides and the MS/MS fragments 27 ., The distances between crosslinked residues are based on the length of the crosslinker reagents , and were introduced as constraints into the DMD potentials ( see section below and 11 for additional details ) ., A total of 30 crosslinking constraints were used in these DMD simulations ( S1 Table ) ., In addition , α-synuclein was characterized by top-down ECD- and UVPD-FTMS HDX and CD to determine the secondary-structure content ( Fig 1 and S1 Fig ) ., Quantitative differential surface modification experiments were performed with and without 8 M urea to determine the characteristics of the residues as exposed or buried ( S2 Table ) ., LD-CL was used to estimate the overall protein topology ( S3 Table ) ., α-Synuclein was expressed using a pET21a vector provided by Dr . Carol Ladner of the University of Alberta ., The protein was expressed in E . coli BL21 ( DE3 ) bacteria and was purified as in 25 ., Briefly , the protein was overexpressed with 1 mM IPTG in 1L LB cultures of BL21DE3 E . coli for 4 hours at 30°C ., Cells were lysed with a French press and the lysate was heated at 70°C for 10 minutes and then centrifuged at 14000 g for 30 minutes ., The soluble fraction was precipitated for 1 hour in 2 . 1 M ( NH4 ) 2SO4 ., α-Synuclein was then purified by fast protein liquid chromatography on a Mono Q 4 . 6/100 SAX column ( GE Life Science ) , using a gradient from 50–500 mM NaCl , in 50 mM Tris at pH 8 . 0 ., Elution fractions containing α-synuclein were further purified by size exclusion on a Superdex 200 30/100 GL column ( GE Life Science ) ., For the expression of metabolically labeled 15N α-synuclein , 1L of M9 Minimal media was prepared with 1 g/L 15NH4Cl ( Cambridge Isotopes ) as the sole source of nitrogen ., BL21 ( DE3 ) cells were grown overnight in 50mL of this media , then seeded into 1 L , grown to an A600 of approximately 0 . 8 , and induced using 1 mM IPTG ., After expression overnight at 30°C , 15N α-synuclein was purified as described above ., Unlabeled and 15N metabolically-labeled α-synuclein were mixed in a 1:1 ratio at a concentration of 20 μM in 50 mM Na2HPO4 and incubated overnight at room temperature prior to crosslinking ., α-synuclein aliquots of 38 μL were then crosslinked using either 1 mM of the ABAS-12C6/13C6 crosslinker ( Creative Molecules ) or 30 mM of the EDC crosslinker ., ABAS crosslinking reaction mixtures were incubated for 10 minutes in the dark to allow the NHS-ester reaction to take place , followed by 10 minutes of UV irradiation under a 25 W UV lamp ( Model UVGL-58 Mineralight lamp , UVG ) with a 254 nm wavelength filter ., ABAS reaction mixtures were quenched with 10 mM ammonia bicarbonate ., EDC reaction mixtures were incubated for 20 minutes ., A portion of each crosslinking reaction mixture was checked by SDS-PAGE gel to see the extent of potential intermolecular crosslinked products ., Aliquots were subsequently split and digested with either trypsin or proteinase K at an enzyme: protein ratio of 1:10 ., Digestion was quenched using a final concentration of 10 mM AEBSF ( ApexBio ) , and samples were then acidified with formic acid for analysis by mass spectrometry ., For TATA , 100 μM synuclein in 50 mM sodium phosphate buffer was reacted with 0 . 5 mM TATA-12C3/13C3 ( Creative Molecules ) ., Samples were incubated for 5 minutes with 254 nm UV light from the same lamp as was used for the ABAS reactions ., Samples were then split and digested with either proteinase K or trypsin at an enzyme: protein ratio of 1:20 ., For SDA reactions , 20 μL of 1mg/mL α-synuclein was crosslinked using 1 mM SDA ( Creative Molecules , Inc . ) ., Aliquots were incubated for 15 minutes in the dark prior to incubation under the same UV lamp as used previously for ABAS reactions but changing the wavelength to 366 nm ., Samples were then run on an SDS-PAGE gel , and bands representing the α-synuclein monomer were excised and subjected to in-gel trypsin digestion ., After in-gel digestion , samples were acidified using formic acid prior to mass spectrometric analysis ., The CBDPS crosslinking reaction mixture consisted of 238 μL of 50 μM α-synuclein , with 0 . 12 mM CBDPS ., Samples were split and digested with either proteinase K or trypsin at an enzyme: protein ratio of 1:10 ., Digests were quenched with 10 mM AEBSF and samples were enriched using monomeric avidin beads ( Thermo Scientific ) ., Enriched samples were acidified for mass spectrometric analysis using formic acid ., Mass spectrometric analysis was then performed using a nano-HPLC system ( Easy-nLC II , ThermoFisher Scientific ) , coupled to the ESI-source of an LTQ Orbitrap Velos or Fusion ( ThermoFisher Scientific ) , using conditions described previously 11 ., Briefly , samples were injected onto a 100 μm ID , 360 μm OD trap column packed with Magic C18AQ ( Bruker-Michrom , Auburn , CA ) , 100 Å , 5 μm pore size ( prepared in-house ) and desalted by washing with Solvent A ( 2% acetonitrile:98% water , both 0 . 1% formic acid ( FA ) ) ., Peptides were separated with a 60-min gradient ( 0–60 min: 4–40% solvent B ( 90% acetonitrile , 10% water , 0 . 1% FA ) , 60–62 min: 40–80% B , 62–70 min: 80% B ) , on a 75 μm ID , 360 μm OD analytical column packed with Magic C18AQ 100 Å , 5 μm pore size ( prepared in-house ) , with IntegraFrit ( New Objective Inc . , Woburn , MA ) and equilibrated with solvent A . MS data were acquired using a data-dependent method ., The data dependent acquisition also utilized dynamic exclusion , with an exclusion window of 10 ppm and exclusion duration of 60 seconds ., MS and MS/MS events used 60000- and 30000-resolution FTMS scans , respectively , with a scan range of m/z 400–2000 in the MS scan ., For MS/MS , the CID collision energy was set to 35% ., Data were analyzed using the 14N15N DXMSMS Match program from the ICC-CLASS software package 27 ., SDA crosslinking data was analyzed using Kojak 28 and DXMSMS Match ., For scoring and assignment of the MS/MS spectra , b- and y-ions were primarily used , with additional confirmation from CID-cleavage of the crosslinker where this was available ., Chemical surface modification with pyridine carboxylic acid N-hydroxysuccinimide ester ( PCAS ) ( Creative Molecules ) was performed as described previously 29 ., Briefly , α-synuclein was prepared at 50 μM in 8 M urea in PBS , pH 7 . 4 ( unfolded state ) , or in only PBS ( folded state ) ., Either the light or the heavy form of the 13C-isotopically-coded reagent ( PCAS-12C6 or PCAS-13C6 ) was then added to give a final concentration of 10 mM ., Reaction mixtures were incubated for 30 minutes and quenched with 50 mM ammonium bicarbonate ., Samples were then mixed at a 1:1 ratio , combining folded ( PCAS-12C ) with unfolded ( PCAS-13C ) samples , as well as in reverse as a control ., Samples were acidified with 150 mM acetic acid and digested with pepsin at a 20:1 protein: enzyme ratio overnight at 37°C ., After digestion samples were prepared for mass spectrometry analysis using C18 zip-tips ( Millipore ) ., Zip-tips were equilibrated with 30 μL 0 . 1% TFA , sample was introduced , then washed with 30 μL 0 . 1% TFA and eluted with 2 μL of 0 . 1% formic acid/50% acetonitrile ., Samples were analyzed by LC-MS/MS as described above ., Top-down ECD-FTMS hydrogen/deuterium exchange was performed as described previously 30 ., Briefly , protein solution and D2O from separate syringes were continuously mixed in a 1:4 ratio ( 80% D2O final ) via a three-way tee which was connected to a 100 μm x 5 cm capillary , providing a labeling time of 2 s ., The outflow from this capillary was mixed with a quenching solution containing 0 . 4% formic acid in 80% D2O from the third syringe via a second three-way tee and injected into a Bruker 12 T Apex-Qe hybrid Fourier Transform mass spectrometer , equipped with an Apollo II electrospray source ., In-cell ECD fragmentation experiments were performed using a cathode filament current of 1 . 2 A and a grid potential of 12 V . Approximately 800 scans were accumulated over the m/z range 200–2000 , corresponding to an acquisition time of approximately 20 minutes for each ECD spectrum ., Deuteration levels of the amino acid residues were determined using the HDX Match program 31 ( S1 Fig ) ., Synuclein UVPD spectra were collected on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with a 2 . 5-kHz repetition rate ( 0 . 4 ms/pulse ) 213 nm Nd:YAG ( neodymium-doped yttrium aluminum garnet ) laser ( CryLas GmbH ) with pulse energy of 1 . 5 ± 0 . 2 μJ/pulse and output power of 3 . 75 ± 0 . 5 mW for UVPD ., The solution was exchanged with deuterium using the same three-way tee setup , although in this case a 50 μm x 7cm capillary provided a labeling time of ~1s ., Spectra were acquired for 8 or 12 ms , and resultant spectra were averaged and used for the data analysis with the HDX Match program as above ., CD spectra were recorded on Jasco J-715 spectrometer under a stream of nitrogen ., The content of α-helical and β-sheet structures was calculated using BeStSel web server 32 ., Crosslink guided discrete molecular dynamics ( CL-DMD ) simulations were performed according to the protocol described in our previous work 11 ., Briefly , discrete molecular dynamics ( DMD ) is a physically based and computationally efficient approach for molecular dynamics simulations of biological systems 16 , 17 ., In DMD , continuous inter-atom interaction potentials are replaced with their discretized analogs , allowing the representation of interactions in the system as a series of collision events where atoms instantaneously exchange their momenta according to conservation laws ., This approach significantly optimizes computations by replacing integration of the motion equations at fixed time steps with the solution of conservation-law equations at event-based time points 33 ., In order to incorporate experimental data for inter-residue distances between corresponding atoms into DMD simulations , we introduced a series of well-shape potentials that energetically penalize atoms whose interatomic distance do not satisfy experimentally determined inter-atom proximity constraints ., The widths of these potentials are determined by the cross-linker spacer length and side chain flexibility 11 ., Starting from the completely unfolded structure of α-synuclein molecule , we performed an all-atom Replica Exchange ( REX ) 34 simulations of the protein where 24 replicas with temperatures equally distributed in the range from 0 . 375 to 0 . 605 kcal/ ( mol kB ) , are run for 6 x 106 DMD time steps ( S2 Fig ) ., The simulation temperature of each of the replicates periodically exchanged according to the Metropolis algorithm allowing the protein to overcome local energetic barriers and increase conformational sampling ., During the simulations we monitored the convergence of the system energy distribution specific heat curve , calculated by Weighted Histogram Analysis Method ( WHAM ) 35 which was used as the indicator of system equilibration ., We discarded the first 2 x 106-time steps of system equilibration during the analysis ., Next , we ranked all of the structures among all of the trajectories , and selected the ones with lowest 10% of the energies , as determined by the DMD Medusa force field 36 ., These structures were then clustered using the GROMACS 37 distance- based algorithm described by Daura et al . 38 ., It uses root-mean-square deviation ( RMSD ) between backbone Cα atoms as a measure of structural similarities between the cluster representatives ., A RMSD cut-off was chosen to correspond to the peak of the distribution of pair-wise RMSDs for all of the low-energy structures ., Because the energies of the resulting centroids representative of the clusters are very close to each other ( S3 Fig ) and picking one of them would potentially introduce a bias related to our scoring energy function , we presented them all as our predicted models of the α-synuclein globular structure ., We then calculated the root-mean-square deviation of atomic positions within each cluster and used this as a measure of fluctuations of the structures of corresponding centroids ( Figs 2 and 3 ) ., In order to obtain information on the global folding of α-synuclein , we performed clustering analysis on the lowest-energy structures obtained during CL-DMD simulations ., In summary , we have determined de novo the conformational ensemble of native α-synuclein in solution by short-distance crosslinking constraint-guided DMD simulations , and validated this structure with experimental data from CD , HDX , SM , and LD-CL experiments ., The predicted conformational ensemble is represented by rather compact globular conformations with transient secondary structure elements ., The obtained structure can serve as a starting point for understanding the mis-folding and oligomerization of α-synuclein .
Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction ., Here , we apply a recently-developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations ( CL-DMD ) for the determination of conformational ensemble of the intrinsically disordered protein α-synuclein in the solution ., The predicted structures were in agreement with hydrogen-deuterium exchange , circular dichroism , surface modification , and long-distance crosslinking data ., We found that α-synuclein is present in solution as an ensemble of rather compact globular conformations with distinct topology and inter-residue contacts , which is well-represented by movements of the large loops and formation of few transient secondary structure elements ., Non-amyloid component and C-terminal regions were consistently found to contain β-structure elements and hairpins .
As the population ages , neurodegenerative diseases such as Parkinson’s disease will become an increasing problem in many countries ., Aggregation of the protein α-synuclein is the primary cause of Parkinson’s disease , but there is still a dearth of structural information pertaining to the native , non-aggregating form of this protein ., A better understanding the structural state of the native protein may prove useful for the design of new therapeutics to combat this disease ., In order to obtain more structural information on this protein , we have recently modelled the native α-synuclein protein ., These models were generated using a novel approach which combines protein crosslinking and discrete molecular dynamics simulations ., We have found that the α-synuclein protein can adopt several shapes , all with a similar topology , resembling a three fingered closed claw ., A region of the protein important for aggregation was found to be protected from the surrounding biological environment in these conformations , and the stabilization of these structures may be a fruitful avenue for future drug research into mitigating the cause and effect of Parkinson’s disease .
chemical bonding, molecular dynamics, protein structure prediction, protein structure, intrinsically disordered proteins, physical chemistry, protein structure determination, proteins, chemistry, cross-linking, molecular biology, protein structure comparison, biochemistry, biochemical simulations, biology and life sciences, physical sciences, computational chemistry, computational biology, macromolecular structure analysis