Energy Landscapes of Human Acetylcholinesterase and Its Huperzine

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The Energy Landscapes of human Acetylcholinesterase and its Huperzine A-Inhibited Counterpart Marcus Trapp, Marie Trovaslet, Florian Nachon, Michael Marek Koza, Lambert van Eijck, Flynn Hill, Martin Weik, Patrick Masson, Moeava Tehei, and Judith Peters J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp304704h • Publication Date (Web): 27 Nov 2012 Downloaded from http://pubs.acs.org on November 27, 2012

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The Journal of Physical Chemistry

The Energy Landscapes of Human Acetylcholinesterase and its Huperzine A-Inhibited Counterpart Marcus Trapp,†,4 Marie Trovaslet,k Florian Nachon,k Marek M. Koza,§ Lambert van Eijck,§ Flynn Hill,⊥ Martin Weik,† Patrick Masson,k Moeava Tehei,⊥ and Judith Peters∗,† Comissariat à l’Energie Atomique, Institut de Biologie Structurale, F-38054 Grenoble, France, Centre national de la recherche scientifique, UMR5075, F-38027 Grenoble, France, Université Joseph Fourier, F-38041 Grenoble Cédex 9, France, Institut Laue Langevin, F-38042 Grenoble Cédex 9, France, Institut de Recherche Biomédicale des Armées Antenne de La Tronche, France, School of Chemistry and Centre for medical Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia, Université Joseph Fourier, F-38041 Grenoble Cédex 9, France , and Australian Institute of Nuclear Science and Engineering (AINSE), Menai, NSW, Australia E-mail: [email protected]

∗ To

whom correspondence should be addressed à l’Energie Atomique, Institut de Biologie Structurale, F-38054 Grenoble, France ‡ Centre national de la recherche scientifique, UMR5075, F-38027 Grenoble, France ¶ Université Joseph Fourier, F-38041 Grenoble Cédex 9, France § Institut Laue Langevin, F-38042 Grenoble Cédex 9, France k Institut de Recherche Biomédicale des Armées Antenne de La Tronche, France ⊥ School of Chemistry and Centre for medical Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia # Université Joseph Fourier, F-38041 Grenoble Cédex 9, France @ Australian Institute of Nuclear Science and Engineering (AINSE), Menai, NSW, Australia 4 Current address: Applied physical chemistry, University of Heidelberg, 69120 Heidelberg, Germany and Helmholtz Zentrum Berlin, 14109 Berlin, Germany † Comissariat

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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

Abstract Enzymes are animated by a hierarchy of motions occurring on time scales that span more than 15 orders of magnitude from femtoseconds (10−15 s) to several minutes. As a consequence, an enzyme is characterized by a large number of conformations, so-called conformational substates that interconvert via molecular motions. The energy landscape of these macromolecules are very complex and many conformations are separated by only small energy barriers. Movements at this level are fast thermal atomic motions occurring on a time scale between 10−7 and 10−12 s, which are experimentally accessible by incoherent neutron scattering techniques. They correspond to local fluctuations within the molecule and are believed to act as coupling links for larger, conformational changes. Several questions related to this hierarchy of motions are a matter of very active research: which of the motions are involved in the biological functions of the macromolecule and are motions of different energy(and thus time-) scale correlated? How does the distribution of motions change when an enzyme is inhibited? We report here on investigations of the enzyme human acetylcholinesterase, unliganded and in complex with the non-covalent inhibitor Huperzine A, by incoherent neutron scattering. Different time scales are explored to shed light on the interplay of enzyme activity, dynamics and inhibition. Surprisingly the average molecular dynamics do not seem to be altered by the presence of the inhibitor used in this study within the considered time scales. The activation energy for the free and the inhibited form of the enzyme is moreover found to be almost identical despite changes of interactions inside the gorge, that leads to the active site of the enzyme. Keywords: incoherent neutron scattering, molecular dynamics simulations, human acetylcholinesterase, huperzine A, protein inhibition, protein stability

Introduction We are used to see macromolecules as colorful ribbon diagrams that are based on the static structures determined e.g. by X-ray crystallography. Yet, the seemingly static structures are in reality animated by a multitude of motions that bring them to life. Neutrons constitute a particularly use2 ACS Paragon Plus Environment

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ful probe to investigate protein molecular dynamics (for an overview see e.g. 1 ). Neutrons have energies of the order of meV and wavelengths comparable to fluctuation amplitudes of biological samples (in the order of Å). As neutrons carry no electric charge, they do not interact with charged particles such as e.g. electrons but with the nuclei of the sample. Scattered intensity can be divided into two parts: a coherent part which results from the interference of waves scattered by different nuclei and which informs on structure and collective dynamics, and an incoherent part which is scattered from the same nucleus at different times giving thus access to information on dynamics of the sample. The incoherent neutron scattering intensity is dominated by the signal arising from hydrogens. This is due to the hydrogen incoherent scattering cross section, which is one order of magnitude larger than that of all other elements usually occurring in biological matter, and also of its isotope deuterium. 2 The technique thus probes average protein dynamics because hydrogen atoms are almost uniformly distributed in proteins. Cholinesterases (ChEs) are serine hydrolases with a Glutamic acid/Histidine/Serine (Glu/His/Ser) catalytic triad located at the bottom of a deep active site gorge and fundamental for cholinergic functioning of the nervous system. 3–5 Acetylcholinesterase (AChE), one of the two ChEs found in humans, hydrolyses the neurotransmitter acetylcholine, thereby regulating the concentration of the transmitter at the synapse. Blockade of acetylcholine-mediated neurotransmission is lethal. The second cholinesterase, butyrylcholinesterase (BChE), has no known physiological function. However, it detoxifies poisoning esters and has been proposed as a bioscavenger of nerve agents or in mutant forms, as therapeutic treatment of cocaine overdose. 6,7 Gabel et al. 8,9 published a comparative neutron scattering study of native human BChE and its soman conjugate. Soman is an organophosphorus compound acting as an irreversible inhibitor. Soman phosphonylates the catalytic Ser of BChE. Subsequently the pinacolyl group of soman is dealkylated, leading to methylphosphonyl enzymes. This conjugate is called "aged" enzyme. In aged BChE, in addition to the covalent bond to the catalytic Ser, an oxygen atom of the methylphosphonyl group forms two hydrogen bonds with the oxyanion hole, while the other forms a salt bridge with the catalytic His. An aged enzyme is much more stable than the native one. 10 Such interac-



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tions were thought to have an impact on the whole dynamics of the enzyme; but atomic mean square displacements (MSDs) of native and soman-conjugated BChE were found to be identical below the thermal denaturation temperature of the native enzyme, indicating a common energy landscape. However, the denaturated state of the enzyme showed bigger MSDs compared to the inhibited form, consistent with entropic stabilization of the unfolded state. From these results one could conclude that the stability of the BChE methylphosphonylated by soman was larger that that of its wild type form due to a decrease in entropy. 8,9 A methylphosphonyl group has a relatively limited molecular surface print (80 Å2 Van der Waals surface calculated with the Chimera software 11 ), and it remains to be determined if a ligand with a larger surface of interaction with the active site, could have a measurable effect on MSDs. Recently we showed that human AChE (hAChE) has significantly larger MSDs than hBChE between 200 and 300 K, 12 demonstrating that hAChE is much more flexible than hBChE. Therefore the effect of ligand binding was expected to be more pronounced in the MSDs of hAChE than of hBChE. Thus we decided to investigate hAChE in complex with a tight reversible inhibitor to gain more insight into the relation between ligand binding and local thermal motions, which are typically occurring on time scales covered by neutron spectrometers. In contrary to irreversible inhibitors, reversible ones bind only through weak interactions to an enzyme. It follows that the molecule can readily dissociate from the enzyme so that the level of reversible inhibitor-enzyme complex depends on the dynamical equilibrium between association and dissociation. We looked for ligand candidates among relatively large reversible inhibitors of hAChE. Fasciculin, a three-fingered snake toxin, was a candidate of choice as it binds tightly to hAChE. Moreover, it has a surface of interaction of 1100Å2 with hAChE, fully blocking the entrance of the active site gorge. 13–15 However, owing to the cost of fasciculin, the amount of toxin needed to perform a neutron scattering study makes the experiment prohibitive. A second candidate, (-)-Huperzine A (HupA, figure 1), is a naturally occurring alkaloid isolated from the chinese medicinal herb Huperzia serrata. 16 It is a powerful reversible inhibitor of hAChE with an inhibition constant Ki = 40 nM. 17 Its molecular surface is about 280Å2 , thus an intermediate surface value between the


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surface of interaction of fasciculin and the molecular surface of a methylphosphonyl group. The crystal structure of Torpedo californica TcAChE complexed with HupA revealed that HupA is fully enclosed in the active site gorge. 18 HupA forms multiple interactions including hydrogen bonds between the ligand, the protein and structural water molecules, and important interactions with aromatic and hydrophobic residues. TcAChE and hAChE have a high structural identity (about 53%) with a very low root mean square deviation (about 1 Å). The peptide sequences are 54% homologous among the species of ChEs, and even more for the catalytic subunit. 19,20 There are 14 aromatic residues that line the active site gorge of TcAChE and determine its narrow path. The volumes of the active sites of hAChE and TcAChE are similar and the 14 aromatic residues in the active site gorge of hAChE are also conserved. 21 Tara et al. compared root-mean-square atomic fluctuations of free AChE from mouse and complexed with HupA by 1-ns molecular dynamic simulation (MD). 22 Although differences between the two simulations were small, they proposed that binding of HupA could modify cholinesterase molecular dynamics by decreasing the enzyme global flexibility. To complete our neutron studies we also performed biochemical characterization and MD simulations on dimeric hAChE complexed or not with HupA. In order to observe an as wide as possible time window, we combined elastic incoherent neutron scattering (EINS) experiments on three instruments at the Institut Laue Langevin (ILL), including four different energy resolutions. This approach allowed the observation of motions within time domains ranging from a few tens of picoseconds up to about one nanosecond, corresponding to movements from very fast internal motions to slower global motions. The change of elastic intensity as a function of temperature was recorded. Assuming a Gaussian distribution 23 of the atoms around their equilibrium positions permitted the extraction of the MSDs. They are the reflection of the sampling of conformational sub-states and of vibrational amplitudes (see figure 2), in presence or not of the inhibitor.



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Experimental section Characterisation of recombinant humanAChE Recombinant hAChE was expressed in CHO cells and purified from cell culture medium as previously described. 12 hAChE was concentrated to about 13 mg/ml using a Centricon-30 ultrafiltration microconcentrator (30,000 MW cutoff from Amicon (Millipore, USA)). Enzyme concentration was determined from its absorbance at 280 nm using a molar extinction coefficient of 1.7 for 1 mg/ml of protein. 24 Titration of hAChE was performed using O-n-butyl-S-[2-(diethylamino)ethyl] methylphosphonothioate (VX) concentration from 2 to 8 nM in 0.1 M phosphate buffer pH 7.4 containing 1 mg/ml bovine serum albumin. Inhibition of 10 nM hAChE was performed using a concentration of HupA (1 mg/ml stock solution in 0.1 M HCl) ranging between 2.75 and 11 nM. Residual activity of a 30 µl aliquot was monitored after different incubation times. Activity measurements were carried out at 25◦ C according to the Ellman method 25 using 1 mM acetylthiocholine (ATC) as substrate and 0.5 mM 5-5’-dithio-bis (2-nitrobenzoic acid) (DTNB). Analysis of the kinetic data was performed with ENZO 26 allowing association (kon ) and dissociation (ko f f ) constant determination. Melting temperatures (Tm ) of free and HupA-conjugated hAChE were determined in triplicate by fluorescence-based thermal shift assay as described in. 27 Circular dichroism (CD) spectra of hAChE (0.1 mg/ml in ammonium acetate buffer 5 mM pH 7) were collected on a JASCO-810 CD spectrometer in the spectral range 190-240 nm (at 0.5 nm intervals) at room temperature. Baseline was performed with ammonium acetate buffer 5 mM pH 7. The spectra analyses were performed using the DichroWeb server (Dichro). 28,29

Sample preparation for neutron scattering A batch of about 300 mg of hAChE was dialyzed against 25 mM ammonium acetate dissolved in D2 O, pD 7.0 (corresponding to a pH 6.6, because heavy water is less dissociated and a weaker acid than ordinary water). About half of this solution was mixed with 2 molar equivalents of (-)6 ACS Paragon Plus Environment

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Huperzine A, the naturally occurring enantiomer (Sigma), before lyophilization. In such a way, a saturation of the enzyme was made sure, the additional HupA molecules being in excess. Since the buffer is completely volatile, a 12 hours lyophilization at 220 K under vacuum resulted in salt free protein powder. The second half of the hAChE solution was lyophilized without the inhibitor. Both lyophilized powders (free hAChE and hAChE/HupA complex) were placed in aluminium sample containers of dimensions 30 × 40 × 1 mm3 to match the size of the neutron beam available on the instruments. The samples were dried for 12 hours at atmospheric pressure over P2 O5 and weighed. The measured weights were taken as their dry weights (h = 0 g D2 O/g dry powder, denoted by g/g in the following). For neutron experiments, samples were hydrated by vapour exchange over pure D2 O, at ambient temperature, in a dessicator. A final water content of 0.4 g/g for both samples was achieved, corresponding to at least one full hydration layer at the protein surface. To verify that no loss of material had occurred and that the hydration state was the same, both samples were weighed before and after the neutron scattering experiments. No losses were detected for any sample.

Elastic incoherent neutron scattering The free form of hAChE contains a high proportion of hydrogen, 4673 of a total of 9470 atoms. The incoherent cross section of the hydrogen atoms thus corresponds to 99.8% of the total incoherent cross section and to 92.6% of the total scattering of the sample (without the D2 O hydration layer). Since HupA contains only 18 hydrogen atoms (hence 36 hydrogen atoms for 2 molar equivalents) compared to the 4673 H-atoms of hAChE, the measured signal is largely dominated by the signal of the enzyme, even in the presence of the ligand. The hydrogen content was calculated from the crystal structures, hence no H/D exchange with the buffer was taken into account. A neutron scattering experiment with angular and energy resolution measures the double differential scattering cross section

d2 σ dΩdE ,

which is the number of neutrons scattered per second into the

0 solid angle dΩ in the direction of the scattering vector k~ with an energy in the interval between E

and E+dE, normalized by the incident neutron flux Φ. It can be split into two parts: the coherent and the incoherent scattering. Coherent scattering carries information on the structure of a material 7 ACS Paragon Plus Environment


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and on the collective dynamics of the atoms, whereas incoherent scattering contains information on the average over individual atomic dynamics. It can be interpreted as a superposition of neutron waves that were scattered from the same nucleus at different times. The experimentally accessible time range is determined by the energy resolution of the instrument. The double differential ~ ω) via scattering cross section is connected to the scattering function S(Q, σcoh k0 ~ σinc k0 ~ d2 σ =N S (Q, ω)coh + N S (Q, ω)inc , dΩdE 4π k 4π k

(1)

where σcoh and σinc are the total coherent and incoherent scattering cross sections. k and k’ ~ are the absolute values of the incoming and scattered wave vectors of the neutron, respectively. Q ~ = k~0 − ~k and ~ω = is the scattering vector defined as Q

~2 02 2 2mn (k − k )

= E 0 − E the energy transfer

between the incident and scattered neutron. For elastic scattering ω = 0 ± ∆E, with ∆E being the elastic energy resolution of the spectrometer. The elastic incoherent scattering function S(Q, 0 ± ∆E) can be simplified using the Gaussian approximation, 23 assuming that the distribution of the atoms around their average position follows a Gaussian distribution, to S (Q, 0 ± ∆E) ≈ exp(−2W),

(2)

1 D E W = Q 2 u2 . 6

(3)

where

The exponential term corresponds to the so-called Debye-Waller factor. As Q approaches zero, the approximation is strictly valid for any motion localized in the length-time window of the spectromD E D E eter, and it holds up to u2 Q2 ≈ 1, where u2 is the average atomic mean square displacement 30,31 around the equilibrium positions of the atoms.


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Instrumental aspects and data analysis The characteristics of the spectrometers employed for this study in terms of incident wavelength, accessible Q-range, detectable length-scale, energy resolution and timescale are outlined in table 1. The timescale can be calculated from the energy resolution using the Heisenberg uncertainty principle or Eq. 21 of Zorn et al. 32 assuming a Gaussian shape for the resolution functions. As the limits of the time window are not sharp, rounded values in between both results are given in table 1. On the cold neutron time-of-flight spectrometer IN6 33 two different wavelengths corresponding to two different energy resolutions of ∆E ≈ 50 and 90 µeV were applied. In this setup the spectrometer is sensitive to motions with associated times of a few ps. IN13 34,35 is the only reactorbased thermal neutron backscattering spectrometer worldwide which results in an intermediate energy resolution of ∆E ≈ 8 µeV, corresponding to about 100ps. Finally IN16 36 is a high resolution cold neutron backscattering spectrometer with an energy resolution of ∆E ≈ 0.9 µeV, corresponding to a time window up to ≈ 1ns and therefore rather sensitive to global motions. The different contributions to atomic motions (lattice and internal molecular vibrations, reorientations, translations) are assumed to be decoupled, because they occur on significantly different time scales, what was also shown by MD simulations 37 and quasi-elastic neutron scattering. 38 In this sense the instruments can be seen as a motion filter to focus on certain dynamics occurring within the window of a given spectrometer. All three spectrometers are indeed sensitive to internal motions, which correspond to MSD values of the order of 1 Å2 . By using D2 O for hydration of the sample powder we focused on internal protein dynamics in the current study. Elastic temperature scans in the range of 20 - 310K were performed on all three spectrometers. In the case of IN6 and IN13 scans were done stepwise with an increased measuring time for 20K (used for normalization). Counting times per point were 15 minutes on IN6 and 1-2 hours on IN13, respectively. On IN16 the scans were done in a continuous way, using a ramp of 0.27K/min between 20 and 180K and of 0.2K/min between 180 and 310K. Transmission values were measured on IN13 and corresponded to 0.94 and 0.95 for the free and the inhibited form of hAChE. 9 ACS Paragon Plus Environment


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Multiple scattering effects were therefore not taken into consideration for the data treatment. In order to obtain the scattered intensities of the sample, scattering from the empty sample holder was subtracted, the data were normalized to the lowest temperature (20K) and absorption correction based on the correction formula of Paalman-Pings coefficients 39 was applied. The complete data reduction was carried out using the LAMP software available at ILL. 40

Determination of atomic mean-square displacements On the backscattering instruments IN13 and IN16 the elastic intensity as a function of Q can be obtained directly from recording the scattered neutrons as a function of scattering angle. As IN6 is a time-of-flight instrument, the whole accessible energy range is measured simultaneously. The elastic data were thus extracted by integrating over an energy range of [-0.11, 0.12] meV for the 90 µeV resolution and of [-0.06, 0.09] meV for the 50 µeV resolution, respectively , and thus covering the entire elastic peak. Whereas at low temperatures intensities decreased linearly over the whole Q-range, a deviation from the Gaussian linear behavior appeared in the plots at higher temperatures (see Electronic Supplementary supporting information, figure 1), consistent with the well-known dynamical transition around 220 K, 41 where non-harmonic atomic motions start to rise. The average mean square displacements can be obtained from the slope of the logarithm of the scattered intensities according to D E ∂lnS (Q, 0 ± ∆ω) . u2 = −3 ∂Q2

(4)

This approach is formally similar to the Guinier approximation in small angle scattering. 42 We extracted MSDs from the Q-ranges where the linearity was clearly visible. According to this constraint, the Q-ranges used for fitting were at IN6 Q = 0.49 - 1.24 Å−1 at 90 µeV resolution and Q = 0.42 - 1.13 Å−1 at 50 µeV resolution, at IN13 Q = 0.52 - 1.67 Å−1 and at IN16 Q = 0.54 - 1.1 Å−1 . D E The criterion for validity of the Gaussian approximation u2 Q2 ≈ 1 was checked a posteriori. It is slightly exceeded at IN16 and IN13 (values up to 2.4), but the data points still show a linear


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Q-dependence in the respective Q-ranges chosen and thus the approximation is justified. 43

Molecular Dynamics Simulations The structure of hAChE was prepared from the crystal structure of the dimeric apoprotein (PDB entry 3lii). The missing residues of the surface loop 490-500 in chain B were reconstructed using the equivalent residues of chain A. The missing surface loop 258-265 was rebuilt by generating and minimizing 50 different conformations of the loop using MODELLER 9v8 44 and selecting the conformation with the lowest energy. Molecular dynamics simulations were carried out using GROMACS 4.5.4 45 and the Amber99sb forcefield. 46 The Lennard-Jones interactions were cut off at 1 nm. The long-range electrostatic interactions were handled using particle-mesh Ewald method for determining long-range electrostatics (10 Å cutoff). Temperature was set to 310 K and was kept constant using a Berendsen thermostat (with a coupling time constant of 0.1 ps). Pressure with a reference value of 1 bar was controlled by a Berendsen barostat (with a coupling time constant of 0.5 ps). To build the initial structure of the hAChE/HupA complex, HupA was manually docked in both monomers by superimposing the structure of AChE to the crystal structure of the complex of HupA and Torpedo californica AChE (pdb entry 1vot), 18 the only structure of AChE ever determined experimentally in presence of HupA (see figure 3). This method is supported by the observation that out of the 26 residues located at 6 Å or less from HupA in TcAChE, all are strictly conserved with identical conformation in hAChE but a phenylalanine replaced by a tyrosine residue (Phe330 -> Tyr337 in hAChE). The network of crystallographic water molecules is also conserved between torpedo and human AChE. These critical water molecules were conserved in the initial models. The topological description of huperzine was built using acpype and the general amber force field. 47 The ligand-free dimer or the complex were immersed in a periodic water box of cubic shape with a minimal distance of 12 Å to any edge and periodic boundary conditions. The box was solvated using the TIP4P solvation model and chloride and sodium counter ions at a concentration of 50 mM were added to neutralize the simulation system. After energy minimization using a 500-step steepest decent method, the system was subjected to equilibration 11 ACS Paragon Plus Environment


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at 310 K and 1 bar for 50 ps under the conditions of position restraints for heavy atoms. Full MD simulation was performed for 10 ns at 310 K, using 2 femtosecond timesteps. All bond lengths were constrained using the LINCS algorithm. 48 Coordinates were saved every 500 steps (every ps). The resulting conformation was optimized by a final 500-step steepest descent minimization. Root mean square deviation for backbone atoms as a function of time was calculated using g_rms included in the GROMACS package (see supporting information, figure 2). Atomic position fluctuations and deviations were calculated for the last 8 ns of the simulation using g_rmsf included in the GROMACS package (see results and discussion).

Results and discussion Biochemical characterization of recombinant human acetylcholinesterase Time dependence of the inhibition of hAChE by HupA is shown in figure 4. Fitting the kinetic data with ENZO 26 allows the determination of the association constant kon = 5.8 ± 0.4 × 106 M−1 min−1 , the dissociation constant ko f f = 0.010 ± 0.001 min−1 and the dissociation constant at dynamic equilibrium KD ≈ 1.7 ± 0.2 nM. Thus, at the very high HupA concentration used during the neutron scattering experiment, i.e. 2 molar equivalents, the binding site of the enzyme is permanently saturated and even if a molecule of HupA dissociates, a new molecule readily replaces it in a few ns. Thermostability of ChEs has been shown to increase in the presence of some inhibitors. 49 Melting temperatures of free and hAChE/HupA complex were determined by the thermofluor method (figure 5). HupA inhibition raises hAChE stability against thermal denaturation by only 2◦ C, from 54.7 ± 0.1◦ C (for free enzyme) to 56.4 ± 0.2◦ C (for hAChE-HupA complex). This is not a significant difference and our results thus suggest that thermostabilities of free hAChE and hAChE-HupA complex are almost the same. Activity and CD measurements were performed after the lyophilization step to characterize the samples before the neutron scattering experiments. hAChE activity was mainly preserved (only 12 ACS Paragon Plus Environment

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20-25% of activity was lost). The CD spectrum of the enzyme performed in 5 mM ammonium acetate buffer (pH 7) indicates the presence of a large amount of alpha-helix structures (≈ 60%) and beta-sheets (≈ 20%), in good agreement with the structure of the folded native enzyme (respectively 48% alpha-helix and 15% beta-sheets from pdb entry 1B41) and with results obtained by Raman spectroscopy. 50,51 As our neutron scattering experiments exceeded the temperature of thermal denaturation during the last runs on IN13, dissolution of the lyophilized enzyme was not possible due to unfolding of the protein. Therefore neither activity nor CD measurements could be performed as post neutron check.

MSDs of hAChE with and without HupA on IN6, IN13 and IN16 In order to compare the results for the different resolutions, we extracted the MSDs from the elastic measurements as described above from data collected by each spectrometer (see figure 6, 7 and supporting information, figure 4). The MSDs increase smoothly and display the well-known dynamical transition, 41 which appears between 180 and 230 K depending on the resolution of the instrument. No differences are visible between our both samples on any instrument (fig. 6, 7 and supporting information, fig 4), e.g. for the time scales between 10 ps and 1 ns and internal motions. Neutron scattering probes the average dynamics of all protons present in the protein. Thus the conclusion can be drawn that HupA binding has insignificant impact on the average dynamics in the ns-ps range of the whole protein below 300K, in agreement with our simulation results (see next section). With a decreasing instrumental energy resolution and thus an increasing time window the MSDs increase continuously by more than a factor of two between the smallest and the largest time window (see figure 7) after the dynamical transition. This trend is in accordance with other similar measurements on different spectrometers 52 and the already mentioned characteristic of the instruments to act as a motion filter. The dynamical transition appears in all four figures (fig. 7 and supporting information, fig 4), but becomes more pronounced with finer instrumental resolution. The corresponding onset of anharmonic motions is shifted from 180 K at IN16 to 230 K for the 13 ACS Paragon Plus Environment


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broadest resolution at IN6, as is shown in figure 7 and supporting information, figure 5. Comparing data taken on IN6 and IN16 with simulations over different timescales Becker et al. 53 demonstrated that the instrumental resolution effect leads to such an apparent shift of the dynamic transition temperature. This means, for example, that slow dynamics being detectable with IN16 at a temperature T1 becomes visible at IN6 at another temperature T2 > T1 . The observed decrease in slope of the MSDs at IN16 (see figures 7 and 8 and supporting information, figures 5 and 6) for temperatures higher than 280 K can also be explained by the different instrumental resolution. 12,53–56 This effect was studied in more details within the framework of the neutron frequency window model, 53 which seemed to be the best in our case. Other models 9,57 were investigated to describe these behaviours, but they were not very successful in our case as the resolution dependency of the MSDs was not taken into account explicitly. The transition observed by a spectrometer depends on the relationship between the timescales of the observed relaxation processes and the timescale accessible by the instrument. Two extreme scenarios can be considered: Firstly, if the characteristic relaxation frequencies lie all within the energy resolution of the instrument, the observed dynamical transition is dependent upon the temperature changes of the corresponding atomic MSDs, thus probing the energy levels of different conformational substates. The difference in energy of the substates corresponds to the free energy difference ∆G, which is provided by the increase in thermal energy. 41 In the second case, local minima with almost the same free energy ∆G are separated by a potential barrier corresponding to the activation energy Ea (see figure 2). The contribution to the mean square displacements stays constant if Ea is in the order of RT. 58 We analysed our data according to the neutron frequency window model, which takes explicitly into account the instrumental resolution. The MSDs can be fitted according to Becker et al. 53 through

D E u2 D E where u2

f ast

FW M

D E and u2

slow

D E = u2

f ast

D E + u2

slow

! ∆ω 2 , 1 − arctan π κFW M

(5)

correspond to fast and slow contributions to the mean square dis14 ACS Paragon Plus Environment

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placement, respectively. ∆ω is the half-width at half maximum of the elastic instrumental resolution function and κFW M represents the long-time relaxation frequency corresponding to the D E characteristic time scale of the underlying process. u2 is assumed to depend linearly on temf ast D E perature as u2 = αT and can be obtained by fitting the data at low temperatures with a straight f ast D E line. u2 is a fit parameter taking into account slow diffusive processes. The values for the slow

instrumental resolution functions ∆ω can be found in table 1. Assuming an Arrhenius behaviour for κFW M , it can be expressed as:

κFW M (T ) = ae−Ea /RT ,

(6)

where a is a prefactor, Ea the activation energy, R the ideal gas constant and T the absolute temperature. Typical fits for the IN16 data are shown in figure 8 and supporting information, figure 6, and the results are summarized in table 2. The model takes correctly into account the above mentioned particularities: the shift of the dynamical transition temperature with instrumental resolution, the kink of the IN16 data at 280K and the slow increase of the fast motions at low temperature with increasing time window. The parameter a was fixed to a value characteristic for vibrational freD E quencies. 59 u2 is sensitive to the highest MSD value reached in an experiment. Its values are slow

reasonable for IN16 and IN13, but are afflicted with high error bars for IN6. The activation energy is increasing with the time window. The difference between the highest and the lowest value is about 2 kJ/mol (if one does not take into account the value for hAChE corresponding to IN6 at 50 µeV, where the error is big, due to restricted beamtime), what is of the same order of magnitude as the thermal energy kB T at physiological temperature. The extracted activation energies Ea can be compared with other values found in literature for proteins such as bovine pancreatic trypsin inhibitor (BPTI) (12.6 kJ/mol) 53 and haemoglobin (7.1 kJ/mol). 60 This indicates that the activation energy can differ by a factor of about 2 between two proteins. Comparing hAChE with hBChE, we found a difference in Ea of about + 3 kJ/mol. 12 The dynamical transition occurs about 50K lower in the case of hAChE and a higher catalytic activity compared to hBChE was found. Thus even a rather small variation in the activation energies has a significant 15 ACS Paragon Plus Environment


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meaning. The interpretation of the relaxation frequency needs, however, special care, as underlined also by Becker et al. 53 The relaxation frequency κFW M is not associated with a specific kind of motion and in eq. (11) of their paper only one diffusive motion is considered. This is certainly an approximation and does not hold for biological samples. Furthermore, the model does not give a reasonable value for the mean square displacements in the limit of an instrument with infinitely small resolution. Nevertheless, this very simple approach helps to see the signature for more motions affecting the time window of IN16 compared to the IN13 and IN6 time windows. As the MSDs on IN16 reach higher values at room temperature compared to the other spectrometers, this is also an indication for a higher molecular flexibility. Indeed, the movements detectable at one specific spectrometer depend not only on the instrumental resolution, but also on the momentum transfer range Q and the sample temperature T. It is very challenging to disentangle the different contributions from elastic scans only. Quasi-elastic and inelastic studies are needed to get a more complete picture of the sample dynamics. Nevertheless, the frequency window model permits already to show that there is clearly a difference concerning the motions observable with elastic scans and thus the energy landscape. With the exception of the results for the 50 µeV resolution on IN6, where the acquisition time was shorter and the fitting parameters have thus large error bars, there is a good agreement between the free and the inhibited samples. These similarities of the activation energies of the unliganded and the inhibited form of hAChE might seem surprising at first glance. Tai et al. 61 have shown by MD simulations that half of the atoms in mouse AChE are participating to the breathing mode which is periodically opening the gorge. By blocking the active site one could expect some effect on molecular dynamics visible at the scale of the whole protein. However, large differences in dynamics may exist between mouse and hAChE despite their strong identity (89% identity), as they exist between hBChE and hAChE. 12 Thus we performed MD simulations on hAChE, free and in complex with HupA. Due to the lack of a HupA/hAChE structure (cf Experimental section), the


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available HupA/TcAChE was used to build the complex.1

Molecular dynamics simulations of hAChE with or without HupA The small rmsf of only 0.4 Å of all atoms suggests that HupA remained firmly trapped in the active site gorges of the dimers during the 10 ns simulation. The active site structure of a monomer corresponding to the last frame of the simulation after energy minimization is represented in figure 3. We observed some differences compared with MD simulations performed on the mouse AChE/HupA complex, 22 most of them related to the absence of some highly conserved crystallographic water molecules in these simulations. For example, the carbonyl oxygen of HupA is H-bonded to Tyr133, but the NH group forms an H-bond with a structural water molecule H-bonded to Glu202 and not directly to Glu202. The amino group is not directly H-bonded to Tyr337 or Asp74, but to water molecules H-bonded to these residues, very similarly to what is observed for huprines in mouse AChE. 62 Other interactions are hydrophobic or van der Waals interactions and involve Trp86, Tyr337, Phe338, His447, Tyr449 and Tyr124. The network of hydrogen bonds prone to proton shuttling from Ser203 to Glu202, through His447, Glu334, Ser229, Glu450 remains stable during the whole simulation. The 180◦ flip of Gly121 observed in the Torpedo californica AChE/HupA crystal structure 21 and during the MD simulation of the mouse AChE/HupA complex 22 occurs early in the present simulation at 100 ps (See supporting information, figure 4). It seems to be driven by the orbital repulsion of the carbonyl of HupA and the mainchain carbonyl of Gly120. Besides, new H-bonds form between Gly120-CO and Ala204-NH, Ser125-CO and Gly121-NH, and the methylene of Gly121 fits perfectly in the groove formed by the two cycles of HupA. Since neutron scattering measures proton dynamics, we analyzed the hydrogen rms fluctuations and deviation for all residues in the protein for the unliganded hAChE and the hAChE/HupA complex. As a reminder, the rms fluctuation relates to the mobility of atoms in a residue around their 1 The crystal structure of recombinant human acetylcholinesterase in complex with huperzine A was released during

the reviewing process of this article under pdb entry 4ey5. No significant difference exists between our starting model and the experimental structure.



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average positions, while the rms deviation for a residue indicates the displacement of the average atomic positions from the initial structure positions. We adopted a conservative position by calculating the rms fluctuations and deviations in a time window from 2 to 10 ns of the simulation (see supporting information, figure 2). The average fluctuation for all hydrogen atoms is 1.12 ± 0.59 Å for the unliganded dimer of hAChE and 1.12 ± 0.60 Å for the HupA complex, hence, no difference between the two simulations is observed. The average deviations from the initial structure were found to be 1.67 ± 1.19 Å for the unliganded enzyme and 1.63 ± 0.97 Å for the HupA complex showing the structural stability of the simulation. For both the unliganded enzyme and the HupA complex, the largest fluctuations and deviations are observed for naturally disordered surface loops, in particular Cys258-Asn265 and Pro492-Pro495 (see supporting information, figure 3). We also analyzed the fluctuations and deviations of hydrogen atoms for the residues located within 5 Å of HupA, as they are expected to be the most affected. Here again we observed no significant difference between the unliganded hAChE and HupA complex (figure 9). In conclusion, MD simulations do not evidence a significant change of hAChE proton dynamics induced by HupA binding and are thus in agreement with neutron MSDs presented above.

Conclusions A series of elastic incoherent neutron scattering experiments were carried out on free and HupA inhibited hAChE at different spectrometers of the ILL to probe the different motions of the enzyme. In contrast to the expectations, no difference in MSDs between the free and the inhibited enzyme was detectable on the different instruments. Thus the results from the neutron scattering experiments suggest, that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. MD simulations of hAChE in the presence or absence of the inhibitor undertaken after the experiments support these results. However, the breathing mode of mAChE has a period of about 4 ns and the MD simulations performed by Tara et al. 63 were carried out over 1ns. Thus breathing motions are possibly visible only as


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collective motions on a longer time scale by inelastic neutron scattering and as single-particle diffusion on a longer time scale by quasi-elastic neutron scattering. To verify this hypothesis further investigations are necessary. The MSDs recorded by each instrument increase continuously with the enlargement of the time window. An apparent shift of the dynamical transition temperature from 230 K at 100 µeV to 180 K at 0.9 µeV as well as a decrease in slope of the MSDs at IN16 for temperatures higher than 280 K can be explained by the effect of the instrumental resolution. We analyzed the experimental data within the frequency window model 53 and obtained activation energy values slightly increasing with the time window. This model was chosen as it takes resolution effects explicitly into account. Moreover, we obtained the same results for the activation energy for the free and the inhibited form of the enzyme. We interpret the activation energy as a response of the enzyme to temperature increase. The increase leads to e.g. a rise of flexibility, a progressive breaking of non-covalent interactions without reaching complete denaturation. These small modifications will finally give rise to the onset of diffusive motions. Even if no differences between hAChE and the HupA/AChE complex was observed, the presence of HupA might have an impact on the dynamics at time scales longer than those observed and simulated.

Acknowledgement We are gratefully acknowledging fruitful discussions with F. Natali, F. Gabel, G. Kneller, G. Zaccai, D. Bicout, the ILL for allocation of beam time and the financing of the DGA under contract number REI n◦ 2009340023, DGA/SSA 08co501 and BioMedef 0 PDH-2-NRBC-3-C-301. Financial support by the DTRA (HDTRA1-11-C-0047) is acknowledged. M. Trapp was supported by a Ph.D. scholarship from the French Ministry for Research and Technology. This work is supported by an AINSE Research Fellowship (M. Tehei) and an AINSE PGRA (F. Hill). M. Tehei acknowledges the financial support from the Access to Major Research Facilities Program which is a component of the International Science Linkages Program established under the Australian 19 ACS Paragon Plus Environment


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Government’s innovation statement, Backing Australia’s Ability.

Supporting Information Available An example for the linear fits of ln I vs. Q2 on IN13, as well as the complementary comparisons of the mean square displacements of hAChE and hAChE/HupA complex are given. A compilation of the mean square displacements of the hAChE/HupA complex for all four measured energy resolutions is shown including fits of the frequence window model to the data. Time dependence of the root mean square deviations, root mean square fluctuations and deviations per residue and the evolution of the distance between Ala204 and Gly120-Gly121 extracted from the MD simulations are given. This information is available free of charge via the Internet at http://pubs.acs.org

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Table 1: Characteristics of the spectrometers used in this study. Spectrometer Wavelength [Å] Accessible Q-range [Å−1 ] Length scale [Å] Resolution FWHM [µeV] Timescale [ps] ∆ω [s−1 ]

IN6 5.12 0.4-2.2 2.9-15.7 90 10 6.836×1010

IN6 5.92 0.4-1.8 3.3-15.7 50 20 3.797×1010


IN13 2.23 0.2-4.9 1.3-31.4 8 100 6.076×109

IN16 6.27 0.02-1.9 3.3-314.2 0.9 1000 7.595×108

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Table 2: Values obtained from fitting formula 4 and 5 to the mean square displacements of AChE and AChE/HupA complex. The parameter a was fixed to a value of 1 × 1012 s−1 . Due to the limited acquisition time on IN6 the free AChE sample elastic data could only be measured up to 270 K in the 50 µeV setup, therefore the obtained fit results show large errors compared to the other data sets.

IN16

IN13

IN6 50µeV

IN6 90µeV

fit parameters AChE AChE/HupA complex 2 −1 α [Å K ] 0.00106 ± 0.00005 0.00100 ± 0.00007 2 2 slow [Å ] 0.83 ± 0.02 0.84 ± 0.03 −1 12 a [s ] 1 × 10 1 × 1012 Ea [kJmol−1 ] 14.20 ± 0.11 14.27 ± 0.16 α [Å2 K−1 ] 0.00083 ± 0.00007 slow [Å2 ] 0.97 ± 0.10 −1 a [s ] 1 × 1012 Ea [kJmol−1 ] 12.55 ± 0.39

0.00074 ± 0.00005 1.18 ± 0.09 1 × 1012 12.88 ± 0.30

α [Å2 K−1 ] 0.00069 ± 0.00012 2 2 slow [Å ] 0.88 ± 0.78 −1 a [s ] 1 × 1012 Ea [kJmol−1 ] 9.40 ± 22.20

0.00078 ± 0.00005 0.99 ± 0.15 1 × 1012 14.46 ± 0.54

α [Å2 K−1 ] 0.00056 ± 0.00004 slow [Å2 ] 4.78 ± 1.80 −1 a [s ] 1 × 1012 Ea [kJmol−1 ] 12.25 ± 0.99

0.00058 ± 0.00004 5.65 ± 1.93 1 × 1012 12.62 ± 0.90



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Figure 1: Chemical structure of nootropic alkaloid, (-)-Huperzine A.


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This material is available free of charge via the Internet at http://pubs.acs.org/.



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Ea

G

Figure 2: One-dimensional cross-section through the high-dimensional energy landscape of a protein showing the hierarchy of protein dynamics and the energy barriers (in red: free enzyme, in blue: enzyme with inhibitor).


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Figure 3: Active site of human acetylcholinesterase complexed with (-)-Huperzine A. This model corresponds to the minimized final pose of a 10-ns molecular dynamic simulation of the complex. (-)-Huperzine A is represented in ball and stick with carbon atoms in cyan, nitrogen atoms in blue and hydrogen in white. Active site residues are represented in sticks, with carbon atom in green, nitrogen atoms in blue and oxygen atoms in red. Water molecules are represented in thin sticks. Hydrogen bonds are represented in black dash lines.



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

Figure 4: Time dependence of the inhibition of hAChE by HupA. Enzyme (10 nM) was inhibited by various HupA concentrations (between 2.75 and 11 nM) and residual activities were measured as a function of time.


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Figure 5: Thermostability of free and hAChE/HupA complex determined by fluorescence-based assay.



1.2 1.0

2

[Å ]

0.8

2

0.6 0.4 0.2 0.0 0

50

100

150

200

250

300

350

temperature [K]

Figure 6: MSDs of hAChE (black squares) and hAChE/HupA complex (red circles), hydrated from D2 O vapour and measured at IN16. For a better visibility error bars are given only for a few data points. They are large at high temperature.

3.0 2.5 2.0 2

1.5

2

[Å ]

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

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1.0 0.5 0.0 0

50

100

150

200

250

300

350

400

temperature [K]

Figure 7: Compilation of unliganded hAChE MSDs measured at IN16 (blue diamonds), IN13 (green triangles) and IN6 (50 µeV: red circles, 90 µeV: black squares).


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1.2 1.0 0.8 2

0.6

2

[Å ]

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


0.4 0.2 0.0 0

50

100

150

200

250

300

350

temperature [K]

Figure 8: Mean square displacements of hAChE on IN16 with corresponding fits according to equation 5 and 6.



1.6

1.4

A

AChE AChE+HupA

1.2

RMSF (Å)

1

0.8

0.6

0.4

W 86 Y1 19 G1 20 G1 21 G1 22 F1 23 Y1 24 S1 25 G1 26 A1 27 L1 30 Y1 33 E2 02 S2 03 A2 04 F2 97 Y3 37 F3 38 Y3 41 W 43 9 H4 47 G4 48 Y4 49

2

3 T8

G8

D7 4

0.2

0

Residues

3

AChE AChE+HupA

B 2.5

2

RMSDEV (Å)

1.5

1

W 86 Y1 19 G1 20 G1 21 G1 22 F1 23 Y1 24 S1 25 G1 26 A1 27 L1 30 Y1 33 E2 02 S2 03 A2 04 F2 97 Y3 37 F3 38 Y3 41 W 43 9 H4 47 G4 48 Y4 49

2

3 T8

D7 4

0.5

G8

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

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0

Residues

Figure 9: Root mean square fluctuations (A) and deviations (B) of active site residues hydrogens with or without HupA. RMSF and RMSDEV values for residues within 5 Å of HupA were obtained by averaging hydrogen fluctuations and deviations over the 10 ns trajectories using g_rms of the GROMACS package. The values for identical active site residues of each monomer were averaged and the standard deviation is shown as error bars.


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Energy Landscapes of Human Acetylcholinesterase and Its Huperzine

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