3D structure of the natural tetrameric form of human butyrylcholinesterase as revealed by cryoEM, SAXS and MD

Biochimie 156 (2019) 196e205

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Research paper

3D structure of the natural tetrameric form of human butyrylcholinesterase as revealed by cryoEM, SAXS and MD* Konstantin M. Boyko a, b, *, Timur N. Baymukhametov b, Yury M. Chesnokov b, Michael Hons c, Sofya V. Lushchekina d, Petr V. Konarev b, e, Alexey V. Lipkin b, Alexandre L. Vasiliev b, e, Patrick Masson f, **, Vladimir O. Popov a, b, Michail V. Kovalchuk b, e a

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky pr. 33, bld. 2, Moscow, 119071, Russia National Research Center, Kurchatov Institute, Akademika Kurchatova pl. 1, Moscow, 123182, Russia c EMBL Grenoble, 71 avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France d Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 ul. Kosygina, Moscow, 119334, Russian Federation e Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” Russian Academy of Sciences, Leninsky pr. 59, Moscow, 119333, Russia f Neuropharmacology Laboratory, Kazan Federal University, 18 Kremlevskaia ul., 48000, Kazan, Russian Federation b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 September 2018 Accepted 24 October 2018 Available online 29 October 2018

Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methodsdsingle-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scatteringdwere implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6 Å cryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT)4-peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers. © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Butyrylcholinesterase Acetylcholinesterase Tetramer cryoEM Molecular dynamics 3D structure

Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; cryoEM, single-particle cryo-electron microscopy; MD, molecular dynamics simulations; OP, organophosphate; PRAD, proline rich attachment domain; SAXS, small-angle X-ray scattering; WAT, tryptophan amphiphilic tetramerization domain. * This work is dedicated to the memory of Bhupendra P. Doctor (1930e2014). * Corresponding author. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky pr. 33, bld. 2, Moscow, 119071, Russia. ** Corresponding author. Neuropharmacology Laboratory, Kazan Federal University, 18 Kremlevskaia ul., 48000, Kazan, Russia. E-mail addresses: [email protected] (K.M. Boyko), [email protected], [email protected] (P. Masson). https://doi.org/10.1016/j.biochi.2018.10.017 0300-9084/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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1. Introduction

2.2. CryoEM

Butyrylcholinesterase (EC. 3.1.1.8; BChE) and acetylcholinesterase (EC. 3.1.1.7; AChE are structurally-related sister enzymes forming cholinesterase family. Both enzymes are serine hydrolases with a similar fold common to a∕b-hydrolases. Although the main physiological role of AChE is to terminate the action of the neurotransmitter acetylcholine, the physiological role of BChE has not been completely established. It was recently shown that plasma BChE hydrolyzes ghrelin, the “hunger hormone”, impacting fat metabolism and emotional behavior [1,2]. However, BChE is of toxicological and pharmacological importance, as it hydrolyzes numerous medicamentous and poisonous esters. In addition, it is a stoichiometric bioscavenger of toxic organophosphates [3,4]. Thus, highly purified human BChE can be administered as a biopharmaceutical to protect AChE against poisoning by OP pesticides and chemical warfare nerve agents [5]. BChE is a template for the design and development of detoxification biocatalysts (catalytic bioscavengers against cocaine and heroin toxicity [2,6] and OPs [7]). BChE is also a sensitive marker for the diagnosis of exposure to OPs [8,9] and a therapeutic target for Alzheimer's disease [10,11]. In human plasma, BChE exists as four molecular forms. The major form of BChE is an oligomer (340 kDa) composed of 4 identical subunits, organized as a dimer of dimers in which each dimer is composed of disulfide-bonded monomers [12,13]. Due to partial proteolysis, a small fraction of the tetramer is clipped from the C-terminal. This clipping leads to the loss of intermonomeric disulfide bonds and generates unstable tetramers, dimers and monomers [14,15]. As for AChE, a polyproline-rich attachment peptide, or PRAD, of ~3 kDa is involved in stabilizing the tetrameric structure through a complex assembly [16]. This PRAD is a part of a processed polyproline protein (lamellipodin) product of the RAPH-1 gene [17]. More than 50 structures of monomeric human BChE have been solved to date, including the apo-enzyme, complexes with different ligands and conjugates with irreversible inhibitors. The reported Xray structures of the truncated 5 sugar-off BChE [18] and natural monomer [19] are close to that of Torpedo california AChE [20] and human AChE [21]. In the past 30 years, all attempts to crystallize the natural tetramer of BChE have failed. These trials included the classical hanging drop approach and crystallization under high hydrostatic pressure and in microgravity conditions. It is assumed that the high sugar content of the enzyme, 25% of its mass, i.e., 9 highly flexible asparagine-linked glycan chains of complex type, impair the proper stacking of molecules and hamper crystallization. Thus, only a modeled structure of the tetrameric human BChE similar to the modeled AChE tetramer [22] has been available so far [23]. Here, we describe the 3D structure of the human BChE tetramer at a 7.6 Å resolution obtained by a combination of cryoEM and MD and finally checked to be consistent with SAXS data. The solved actual tetrameric structure differs notably from the previous model in a number of structural features.

2.2.1. CryoEM sample preparation and data acquisition The stock solution of BChE was diluted in the same buffer to a concentration of 0.3 mg/ml 3.0 ml of the BChE solution were immediately applied to glow-discharged Lacey 300 mesh grids (PELCO NetMesh) that was then plasma-treated for 30 s and 25 mA amperage using a glow discharge cleaning system (PELCO easiGlow) operated under 0.26 mbar pressure of atmospheric gas. The grid was blotted for 1 s without a waiting period and then flash frozen in liquid ethane cooled by liquid nitrogen with a Vitrobot Mark IV (Thermo Fisher Scientific) at 100% humidity and a temperature of 4
C. The sample was imaged at CM01 beam time at ESRF using the EPU (ver. 1.11) software for automated data acquisition and a Titan Krios cryo-electron microscope (Thermo Fisher Scientific) operated at 300 kV with a Quantum LS imaging filter (Gatan). Image stacks were recorded with a K2 Summit (Gatan) direct electron detector operating in counting mode at a recording rate of 4 raw frames per second. The microscope magnification was 130,000 (corresponding to a calibrated sampling of 1.067 Å per pixel). The total dose was 42 electrons per Å2 with a total exposure time of 7 s, yielding 28 frames per stack. A total of 1500 image stacks was collected with a defocus range of 3.0 to 1.0 mm (Table 1).

2. Materials and methods

2.2.2. Single particle image processing and 3D reconstruction Frame alignment and dose weighting were performed using UCSF Motioncor2 [25]. CTF estimation on aligned, unweighted, micrographs was performed with Gctf [26]. All data processing steps except for the particle picking procedure were conducted in RELION 2.1 [27,28]. The BChE particles were picked automatically using Gautomatch software (https://www.mrc-lmb.cam.ac.uk/ kzhang/Gautomatch/) with an automatically generated template. This resulted in 170,963 particles, which were extracted (256  256 pixels) and downsampled (128  128 and 64  64 pixels) for the iterative reference-free 2D classification. In total, 49,261 particles of 2D classes that possessed the quaternary structure features (Fig. 1A) were subjected to 3D classification. A low-resolution initial tetramer model, required for the 3D classification stage, was built in EMAN2 [29] using the corresponding class averages. The 3D classification resulted in five 3D classes, of which only one class had the quaternary structure features characteristic of tetramer. Subsequent 3D refinement of this tetrameric class containing 6712 particles was done without applying symmetry. This subset of particles was used for the final refinement (Fig. 1B). The refinement was made using a soft mask encompassing the whole map and C2 symmetry based on preliminary knowledge of the tetramer structure and led to an improvement of the resolution. The 7.6 Å resolution of the final map was estimated by the 0.143 FSC criterion after a postprocessing procedure (Fig. 1C). Estimation of the local resolution was done with ResMap [30] with the soft mask used in postprocessing (Fig. 1D). The final 3D maps and atomic structures were visualized and analyzed with UCSF Chimera [31], Coot [32] and PyMOL Molecular Graphics System, Version 1.8.6.1 €dinger, LLC (https://pymol.org). The 3D map was deposited in Schro the Electron Microscopy Data Bank (EMDB) under the accession code EMD-0256.

2.1. Protein production The human BChE tetrameric form was highly purified from plasma Cohn fraction IV-4 [24]. The stock solution of tetrameric BChE with a specific activity of 600 units/mg and purity >95% was provided by the late B.P. Doctor (WRAIR, Washington DC, USA). The enzyme at 15 mg/ml was in 25 mM sodium phosphate buffer pH 8.0 containing 1 mM EDTA.

2.2.3. CryoEM model building The X-ray structure of the partially deglycosylated truncated BChE monomer (PDB: 1P0I [18]) was used as the initial model for tetramer building. The C-terminal tails were homology modeled with Modeller 9.14 [33], using the X-ray structure of the AChE tetramerization domain as a template (PDB:1VZJ [22]). Polyproline peptides of 15e18 proline residues were considered, as these

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Table 1 CryoEM data collection and processing statistics. Data collection Accelerating voltage, kV Nominal/Calibrated magnification Pixel sizea, Å Total exposure time, sec Number of stacks Total dose per stack, e/Å2 Number of frames per stack Defocus range, mm Defocus step, mm Data processing Total extracted particles Refined particles Final particles used Symmetry Global resolutionb, Å FSC0.5 (unmasked/masked) FSC0.143 (unmasked/masked) Local resolution rangec, Å EMDB ID a b c

300 130 000/46860. 1.067 7 1500 .42 28 [-3.0; 1.0] 0.4 170963 49261 6712 C2 13.0/9.4 9.1/7.6 [6.2; 8.7] EMD-0256

Calibrated pixel size at the detector. According to RELION 2.1. According to ResMap 1.1.4.

Fig. 1. Quality of the cryoEM data. A e Class averages obtained after 2D classification, subjected to 3D classification. B - Distribution of Euler angles (generated by RELION). С - FSC curves of the masked density maps without imposing symmetry (dotted line) and with C2 symmetry applied (solid line) (according to RELION). D - Local resolution of the final density map estimated with MonoRes, shown on the complete volume (left) and on a coronal cross-section (right).

lengths fit well into the cryoEM density. Loops connecting the enzyme globule with the C-terminal tails were modeled and fitted manually into clear density blobs. Three N-terminal residues and three amino acids located on the surface of the protein are missing in the X-ray structure of the BChE monomer. The coordinates of Asp378 and Asp379 were taken from another PDB X-ray structure of BChE, (PDB:2PM8 [19]), while residues 1e3 and the side chain of Gln455 were reconstructed using the VMD psfgen module. Fitting of the obtained model in the cryoEM density was performed with Chimera and Coot.

2.3. MD simulations 2.3.1. Structure preparation The initial cryoEM model was taken as a starting model for MD simulations. Hydrogen atoms were added to the model with consideration to the hydrogen bonding network using Reduce software [34]. Glycan side chains were restored at the initial glycosylation sites of human BChEdasparagine residues 17, 57, 106, 241, 256, 341, 455, 481, and 486 [35]dand the structures of Nglycans were built according to the structures described in Ref. [36]

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with the help of the Glycoconjugate Data Bank (http://www. glycostructures.jp) and the Glycan Reader & Modeler module of CHARMM-GUI [37,38]. Polyproline peptides of 15e18 amino acids long were used for modeling. The initial position was obtained during homology modeling, with the structure of the AChE tetramerization domain as a template. From this position, polyproline peptides were moved along the principal axis to 1, 2 and 3 Å in both directions and rotated around the axis with a 45
step. A 1 ns MD simulations were performed for all these variants with the BChE Ca atoms constrained. The model with peptides including 17 Pro residues was in the best agreement with the experimental density and was chosen for the further steps. Two forms of C-terminal disulfide bond linking were considered. Details of the construction procedure are provided in the supplementary materials. 2.3.2. Simulations details VMD software [39] was used for system preparation and analysis of results. For all constructed systems, TIP3P water molecules were added, forming a box with boundaries exceeding 10 Å from the glycosylated protein. To make the systems electroneutral, Naþ and Cl ions were added in a final concentration 0.15 M. All systems constructed in this study totaled over 500 000 atoms, and the simulation box size was approximately 200 Å  200 Å  150 Å. Namd 2.12 [40] with a CHARMM36 force field [41,42] was used for all MD simulations. During MD simulations, the systems were maintained at a constant temperature of 298 K under a pressure of -Hoover 1 atm (NPT ensemble) using Langevin dynamics and a Nose barostat. Each MD trajectory for the newly built model described in Supplementary materials was preceded by 2000 steps of energy minimization. Periodical boundary conditions and PME electrostatics were applied. NAMD and VMD implementations of the MDFF method were used [43]. To compare the constructed models and their evaluation along the MD trajectories, the crosscorrelation coefficient (CCC) was calculated. Final model validation was performed with MolProbity [44], and the model fit into the cryoEM density was checked with Chimera. 2.4. SAXS experiments Small-angle X-ray scattering patterns were acquired using standard procedures at the BM29 Bio-SAXS beamline [45] of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at a wavelength of 0.99 Å using a Pilatus1M detector (DECTRIS, Switzerland). The sample-to-detector distance was 2.87 m. The samples were placed in a special thermostatted robotic system [46] in a 200-mL polystyrene cell. The solution from the cell was automatically fed into a continuous-flow quartz capillary 1.8 mm in diameter, in which measurements were carried out. The solution under study was uniformly transmitted through the capillary, and the beam was directed to the same point on the capillary (different sample areas were irradiated at different moments). The data acquisition and preliminary processing (radial averaging, normalization to the transmitted-beam intensity, test for the presence of radiative damage, and detector sensitivity correction) were performed using the SaxsAnalysis pipeline system [47]. Measurements were performed at five different protein concentrations ranging from 0.2 to 2 mg/ml. In total, 10 successive frames with an exposure time of 0.5 s per frame at a constant temperature of 293 K were analyzed, and no evidence of radiationinduced degradation was found. The subsequent data processing was performed with the ATSAS program package [48]. The buffer scattering was subtracted, and the scattering curves measured at different concentrations were averaged and merged using the program PRIMUS [49]. The radius of gyration, Rg, of the protein and the forward

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scattering, I(0), were determined by the Guinier approximation [50] at very small angles (s < 1.3/Rg). Rg was also determined from the entire scattering pattern by the program GNOM [51]. In the latter case, the distance distribution functions p(r) and the maximum particle dimensions Dmax were also computed. The molecular mass (MM) of the solute was calculated by comparing the calculated I(0) value with that of bovine serum albumin (MM ¼ 66 kDa) solution. The excluded volume of the hydrated protein molecule (Vp) was calculated using the Porod approximation [52]. Low-resolution shapes of BChE were produced by the ab initio program DAMMIF [53], a fast version of DAMMIN [54], using P222 symmetry that employs a simulated annealing procedure to build a compact dummy-atoms (beads) model that fits the experimental data, I(s), to minimize the discrepancy. The scattering from the optimized atomic MD models was calculated using the program CRYSOL [55] and compared to the experimental data. Superposition of the ab initio shape envelope and the all-atom MD models was performed using SUPALM [56]. The experimental intensity was also represented by a linear combination of the curves computed from the most populated clusters of conformations from MD trajectories and optimized by OLIGOMER [49]. 3. Results 3.1. CryoEM model of BChE tetramer The dataset was collected using a K2 detector on a Titan Krios electron microscope. Raw particles showed a moderate distribution of the expected tetramers, and reference-free 2D averages (Fig. 1A) clearly demonstrate the presence of the expected oligomeric state. An initial reconstruction without symmetry applied gave a 10.5 Å resolution. C2 symmetry application, together with mask-enforced reconstruction, provided a 9.1 Å resolution, which was finally increased to 7.6 Å after postprocessing (Fig. 1C, Table 1). This confirmed that the tetramer is a dimer of dimers, as previously stated [12,13]. The final cryoEM map exhibited characteristic features of the BChE tetramer: a four-monomer arrangement with well-resolved secondary structural elements, as well as a lefthanded superhelical C-terminal tail which lies in the center of the molecule along the 2-fold axis of the tetramer, perpendicular to its “plane” (Fig. 2A and B). To build an atomic model of tetrameric BChE, first, two identical models of the partially deglycosylated truncated BChE monomer (PDB: 1P0I [18]) obtained at the highest resolution of 2.0 Å were manually fitted into the experimental density as rigid bodies, so that one of the dimers composing the tetramer was constructed. Then, the dimer was duplicated using C2 symmetry to form the tetramer. The C-terminal tail from the crystal structure of the AChE tetramerization domain (PDB: 1VZJ [22]) was homology modeled according to the BChE primary sequence (residues 543e569) [57] and manually fitted as rigid bodies into the experimental density. It is noteworthy that the C-terminal tail obtained in this manner did not fit well into the experimental density, being wider and located outside of the density near its very C-terminal. This required manual bending of the helices composing the tail. Finally, residues forming the long tail, connecting each BChE monomer and the corresponding C-terminus, were manually modeled and fitted into the density, as there were clear density blobs in appropriate positions. The obtained model contained 569 residues for each monomer comprising the tetramer, including the polyproline peptide of 17-residue length. All secondary structural elements fit well within the experimental density, indicating that the monomeric structure does not undergo significant 3D changes upon tetramer formation.

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Fig. 2. CryoEM map of the BChE tetramer. A- Top view of the map, where the 2-fold axis, which coincides with the C-terminal tail of BChE, is perpendicular to the plane of the screen. The map is depicted as a semitransparent gray surface. The BChE tetramer model fitted into the experimental density is shown as a cartoon model and colored by monomers. The polyproline peptide is colored in black and indicated with yellow circle. B - Side view of the map. The representation is the same as that in panel A.

The tetramer size is of approximately 130  170 Å in the “plane” of the monomers and has a “height” of approximately 70 Å, including the C-terminal tail. The interface between the monomers in each dimer is mainly composed of two a-helicesda13 and a18 (Fig. 3) (secondary structural elements numbered according to the ESPript web-server annotation for the human BChE sequence and X-ray structure (http://espript.ibcp.fr)) belonging to each monomer. The monomers composing each dimer could be regarded as related to each other via a 2-fold axis, which goes along and between the a13 and a18 helices of both monomers (Fig. 3). Thus, these monomers are oriented nearly upside down relative to each other, wherein their C-terminals are oriented to properly form the superhelical tail. In the case when the C-terminal tail of the tetramer is oriented vertically in a plane, the monomer arrangement is such that two monomers (upper monomers) from two different dimers are located approximately 15 Å higher than the other two monomers (lower monomers) (Fig. 2B). The C-terminal tail with the clearly seen PRAD peptide has a diameter of 25 Å and a height of

approximately 55 Å. The super coil structure is submerged deep into the tetramer, protruding only approximately 5 Å above the two upper monomers. Such an arrangement of monomers in the tetramer leads to a special orientation of the active site gorges. In the BChE tetramer, the active site gorges of the two monomers belonging to different dimers are oriented on one side of the tetramer, while the active site gorges of the other two monomers are directed to the other side of the tetramer (Fig. 4A and B). For the upper monomers, the distance between the two active site gorges is approximately 80 Å, and they are oriented to the bottom of the tetramer (Fig. 4A). For the lower monomers, the distance between the active site gorges is slightly largerdapproximately 90 Å. These active site gorges are tilted relative to the active site gorges of the upper monomers by approximately 120
and are oriented towards the neighboring upper monomer of the other dimer (Fig. 4B). Such an orientation may have an important consequence, as discussed below. The 9-glycan chains that are linked to asparagine residues of each monomer were almost invisible in the experimental density

Fig. 3. Dimerization interface between two BChE monomers belonging to one dimer. Monomers are shown as cartoons and colored in rainbow manner from the N- to C-terminal. The two other monomers of the tetramer are omitted for clarity. The a13 and a18 helices of each monomer are labeled. The polyproline peptide is colored in black.

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Fig. 4. Active site gorges arrangement in the BChE tetramer. A - Bottom view of the BChE tetramer. Monomers are represented as surfaces colored by chain. Residues forming the active site gorges are shown in yellow, and orientations of the active site gorges are indicated by pink arrows. The polyproline peptide is colored in black. B - Side view of the tetramer. The representation is the same as that in panel A. Note that only one active site gorge for the lower monomers can be visualized, because the other gorge is located on the opposite side of the second lower monomer.

due to its moderate resolution and glycan flexibility. It may be hypothesized, however, that large blobs of density near, for example, residue Asn57 could be attributed to a part of the linked glycan. Therefore, additional effort was required to complement and finalize the BChE structure. 3.2. MD simulations MD simulations were used to construct and optimize the fullatomic model based on the model built from the experimental cryoEM map described above. The simulations of the constructed models showed a high mobility of the glycan chains, explaining their invisibility in the cryoEM density. Such a high glycan mobility required comparison of the most frequent conformations from MD trajectories with SAXS data (see below). The glycans significantly increase the size of the tetramer to approximately 170  220 Å (in the monomer "plane") and to approximately 100 Å along the tetramer's height (Fig. 5A). While monomers in BChE dimers interact through a classical four a-helix bundle, dimer/dimer interfaces are mainly formed by interlaced glycans (Fig. 5A and B). Only in 5% of the snapshots along all the obtained MD trajectories do the residues of the globular part of the different dimers contact directly, mostly via sporadic interactions between Glu255 and Tyr282. Sugar chains swinging over the tetramer surface were found close to the entrance to the catalytic gorge, even obstructing the entrance (Figure S1). Fragments of the sugar chains were found within 4 Å vicinity of the gorge rim (residues Gly333, Ala277, Pro281) in 27.7% of the snapshots. In 3.9% of the snapshots, these sugars enter even deeper into the active site gorge of the monomers, oriented towards the tetramerization domain. Due to such orientation, the sugar chains attached to Asn256 and Asn341, sporadically covering the gorge entrance, interact with the C-terminal (WAT)4/PRAD domain in 85% of snapshots. Good agreement between the full-atomic model and the experimental density was achieved only at the cost of a-helix structure loss from the region linking the globular part to the tetramerization domain (residues 527e535). Interestingly, in this region, a stable interaction between residues Tyr237 and Asp537 of two of the four monomers was observed. This interaction is also clearly seen in the experimental density (Figure S2) and is maintained through the 100 ns MD trajectory, either as a direct hydrogen bond or as an interaction through a bridging water molecule. Additionally, this region is affected by the glycan chain attached to

Asn241, as during MD simulations, this sugar chain is always present in this region. In the homology model of the C-terminal (WAT)4/PRAD based on the AChE tetramerization domain as a template, the very end of each C-terminal is approximately 20 Å away from each neighbor of the (WAT)4/PRAD supercoil, thus protruding from the CryoEM density. This required forced rapprochement between the Cys571 residues of monomers to link the two intermonomeric disulfide bonds of each dimer [12,57]. Experimental data lacks density information for the very end of the C-terminus (residues 569e574). Thus, the exact disulfide bond position cannot be deduced from these data. Therefore, the connection was performed in two variant models, with one of the two neighboring monomers (intradimer disulfide bonds A-B/C-D and interdimer disulfide bonds A-D/B-C variants). After the C-terminal disulfide bonds were linked, the two different models were subjected to MD simulations. The C-termini of the monomers were found to be quite flexible. This outcome may explain the lack of experimental density in this region. Both models are in equally good agreement with the experimental density, suggesting that both modes of connection for disulfide bonding could exist. 3.3. Correlation with the SAXS data To finally validate the different BChE tetramer models, resulting from MD simulations, SAXS data were used. These data were compared to the theoretical scattering patterns calculated from individual MD conformations, representing 20 most populated clusters from MD trajectories, as well as from the planar model of BChE tetramer proposed earlier [23]. The SAXS scattering pattern of BChE is shown in Fig. 6A (black dots with error bars). The radius of gyration and the maximum particle size (Rg ¼ 60 ± 2 Å and Dmax ¼ 222 ± 10 Å), as well as the skewed profile of p(r) with a tail (Fig. 6B), point to the elongated shape of the particle. The molecular mass and the excluded volume of the hydrated protein (MM ¼ 350 ± 30 kDa and Vp ¼ (680 ± 40)  103 Å3) are consistent with the values expected for a tetrameric BChE in solution based on its molecular mass. The Guinier plot (Fig. 6C) shows linear behavior and confirms the sample monodispersity and the lack of aggregates. The restored ab initio shape of the BChE tetramer suggests a structure with a nonplanar monomer arrangement (Figure S3) that neatly fits the experimental data, with c2 ¼ 1.15 (Fig. 6A, red dashed line). The best-optimized MD model of BChE tetramer also provides

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Fig. 5. Full-atomic model of the BChE tetramer obtained after MD, refined into the experimental map. A - Top view of the model. Monomers are depicted as in Fig. 2A. Glycan chains are shown as spherical models. The cryoEM density is shown as a gray semitransparent surface. B - Close-up view of the dimeric interface. The tetramer is shown as a transparent surface and colored by chains. Glycans in the dimeric interface are shown as sticks and have the same color as the corresponding protein chains. Residues Glu255 and Tyr282 are colored in yellow, depicted as sticks and labeled.

Fig. 6. SAXS data. A - SAXS scattering pattern of tetrameric BChE (black dots) as well as calculated curves for the mixture composed of representatives of the most populated conformation clusters from MD trajectory (black solid line), planar model (blue dashed line) and for the ab initio model (red dashed line) are shown. B - The calculated distance distribution function p(r). C - The Guinier plot.

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a good fit (c2 ¼ 1.37) and overlaps well with the ab initio shape of tetramer (Figure S3). The best fit to experimental data (c2 ¼ 1.22) was obtained for the mixture composed of representatives of the most populated conformation clusters from MD trajectory (Fig. 6A, black solid line). At the same time, the "flat" model of BChE tetramer shows a very bad fit with experimental data (c2 ¼ 35.1, Fig. 6A, blue dashed line) and displays systematic deviations from data at low angles. In particular, it cannot adequately reproduce the shoulder observed at angular range of 0.06 Å1< s < 0.10 Å1. This clearly indicates the inconsistency of the "flat" model. 4. Discussion Comparison between the obtained BChE model and the earlierproposed MD model, prBChE [23], revealed a number of discrepancies. First, the prBChE is “flat” so that all four monomers lie in one plane. The experimental BChE model shows a significantly different arrangement of subunits in the tetramer (Fig. 4A and B). Such an arrangement also leads to deep immersion of the C-terminal tail, which is oriented vertically, in contrast to that in prBChE, where the tail protrudes significantly over the monomers and is tilted by 20e45
relative to their plane, depending on the model [23]. Indeed, at the resolution achieved, a discussion about side chain contacts could be only speculative. However, the side chains of a few residues are clearly visible in the density, such as that of tyrosine 237 of two of the four monomers, which form a hydrogen bond to the Asp537 residue of the C-terminal tail. All-atom molecular dynamics simulations allowed optimization of the side chain orientation, as well as building glycan chains on the surface of the tetramer. The obtained model clearly shows that some glycans do participate in the interface between monomers of adjacent dimers (Fig. 5A and B), thus strengthening the tetramer stability in addition to the C-terminal knot. These glycans are linked to asparagine 241 of one monomer and asparagine 256 of the adjacent monomer. At this point, it must be reiterated that the effect of high hydrostatic pressure and subzero temperatures [58], as well as ultrasound [15], on the human BChE tetramer has never shown dissociation of the native unclipped tetramer. This outcome indicates that the majority of noncovalent bonds that stabilize the tetrameric structure are pressure-insensitive. Indeed, according to Le Chatelier's principle, the formation of hydrophobic bonds is accompanied by positive volume changes. Therefore, pressure disrupts hydrophobic bonds. On the other hand, aromatic/aromatic stacking interactions associated with negative volume changes upon formation are favored by pressure. H-bond formation is associated with almost no volume change. H-bonds are therefore pressure-insensitive [59]. This insensitivity strengthens the statement that glycans located at the dimer/dimer interface may play an important role in stabilization of the tetrameric structure. Aromatic-aromatic interactions also play a role in stabilization of the tetrameric structure, in particular at the level of the C-terminal (WAT)4/PRAD. Another interesting feature of the BChE tetramer is the different orientations of the active site gorges. The consequence of this is that glycans attached to residues Asn241, Asn256 and Asn341 could potentially narrow or even cover the entrance of the active site gorges of two of the four monomers in the tetramer (Fig. 5B). Glycan chains attached to Asn256 and Asn341 also frequently interact with the C-terminal (WAT)4/PRAD domain, additionally stabilizing the structure of the tetramer. All other sugar chains are spread over the tetrameric surface and, presumably, have no rigid conformation. A small fraction (approximately 10%) of human BChE was found to be in the form of nonreducible covalent dimers [12,15,60]. These crosslinks were identified as Glu325-Lys267 Asp395-Lys513,

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Glu411-Lys267, Glu460-Lys44, and Glu460-Lys499 [61]. Analysis of the distances between these residues along the MD trajectories of the tetramer showed that in the tetramer, these residues are never in close contact, and the minimum distance between functional groups is 14.8 Å. This outcome suggests that such low-probability crosslinking reactions between side chains occur during the enzyme biosynthesis or folding process. To summarize, we obtained the natural tetrameric structure of human BChE at a 7.6 Å resolution. The structure differs significantly from the models proposed earlier at the level of the quaternary structure, including in the arrangement of monomers and orientation of the PRAD-associated C-terminal tail. To further improve the model, MD simulations were performed. This improvement allowed distinguishing the positions of the asparagine-linked glycans, optimizing side chain orientations and modeling the two intermonomer disulfide bonds at the top of the C-terminal tail. There is a good correlation between the model and the cryoEM data. Finally, the model was successfully checked for consistency with SAXS experimental data, pointing the nonplanar arrangement of the monomers and rejecting the "flat" model proposed earlier [23]. The obtained data provide clear insight into the structural organization of the natural BChE tetramer (Supplementary Video 1). This cryoEM structure is the first example of a PRAD-associated cholinesterase tetramer. Such a unique quaternary structure has only been observed for soluble and synapse membrane-anchored cholinesterases [62]. Knowledge of this quaternary structure will be further useful for developing strategies to improve the conformational stability of the enzyme and its pharmacokinetic properties. These points are essential from the perspective of using human BChE as a bioscavenger for medical countermeasures against OP poisoning or as a biosensor for monitoring anti-cholinesterase agents in the environment. Further improvements in resolution would be helpful for determining the binding/stabilizing roles of glycan chains at the interdimeric interface and possible alternative conformations, mapping unknown allosteric sites and studying conformational changes in the active site gorge when the enzyme is in action. Finally, determination of the cryoEM structure of BChE variants, such as the C5 variant which contains a 60-kDa tail of lamellipodin [17], should provide information on the conformation of the protease target site that leads to processed plasmatic BChE tetramer. Supplementary video related to this article can be found at https://doi.org/10.1016/j.biochi.2018.10.017. Acknowledgement This work was supported by the Russian Science Foundation (project 14-24-00172) as part of the CryoEM data analysis and project 14-13-00124 as part of the molecular modeling. The design and coordination of the research and preparation of the manuscript part for the work, performed by PM, was supported by the Russian Science Foundation (project 17-14-01097). P.V.K. acknowledges the support from the Ministry of Science and Higher Education of the Russian Federation within the State assignment FSRC “Crystallography and Photonics RAS (in part of SAXS measurements). We acknowledge the Resource Center of Probe and Electron Microscopy at the Centre for Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Centre) of the National Research Centre “Kurchatov Institute” for conducting preliminary cryoEM experiments, the European Synchrotron Radiation Facility for provision of beam time on CM01 and BM29 beamlines, Alexander Myasnikov for a fruitful discussion and Alena Nikolaeva for help with the sample preparation for cryoEM experiments. Data processing was done using algorithms developed under the Russian Science Foundation (project 18-41-06001) and computing

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resources of the Federal Collective Usage Center Complex for Simulation and Data Processing for Mega-Science Facilities at NRC “Kurchatov Institute” (ministry subvention under agreement RFMEFI62117X0016), http://ckp.nrcki.ru/. The research was carried out using equipment from the shared research facilities of the HPC computing resources at Lomonosov Moscow State University [63]. The authors acknowledge the Joint Supercomputer Center of the Russian Academy of Sciences for provision of computational time. The authors are grateful to Dr. Vladimir Mironov for technical help. The natural tetrameric human BChE was provided by the late B.P. Doctor (WRAIR, Washington DC, USA), father of cholinesterasebased stoichiometric bioscavengers. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2018.10.017. Compliance with ethical standards Conflict of interests The authors declare no conflict of interest. Human and animal rights This article does not contain any studies with human participants or animals performed by any of the authors. References [1] V.P. Chen, Y. Gao, L. Geng, R.J. Parks, Y.P. Pang, S. Brimijoin, Plasma butyrylcholinesterase regulates ghrelin to control aggression, Proc. Natl. Acad. Sci. U. S. A. 112 (2015) 2251e2256, https://doi.org/10.1073/pnas.1421536112. [2] S. Brimijoin, Y. Gao, L. Geng, V.P. Chen, Treating cocaine addiction, obesity, and emotional disorders by viral gene transfer of butyrylcholinesterase, Front. Pharmacol. 9 (2018) 112, https://doi.org/10.3389/fphar.2018.00112. [3] P. Masson, O. Lockridge, Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior, Arch. Biochem. Biophys. 494 (2010) 107e120, https://doi.org/10.1016/ j.abb.2009.12.005. [4] O. Lockridge, Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses, Pharmacol. Ther. 148 (2015) 34e46, https://doi.org/10.1016/ j.pharmthera.2014.11.011. [5] B.A. Reed, C.L. Sabourin, D.E. Lenz, Human butyrylcholinesterase efficacy against nerve agent exposure, J. Biochem. Mol. Toxicol. 31 (2017), https:// doi.org/10.1002/jbt.21886. [6] K. Kim, J. Yao, Z. Jin, F. Zheng, C.G. Zhan, Kinetic characterization of cholinesterases and a therapeutically valuable cocaine hydrolase for their catalytic activities against heroin and its metabolite 6-monoacetylmorphine, Chem. Biol. Interact. 293 (2018) 107e114, https://doi.org/10.1016/j.cbi.2018.08.002. [7] S.V. Lushchekina, L.M. Schopfer, B.L. Grigorenko, A.V. Nemukhin, S.D. Varfolomeev, O. Lockridge, P. Masson, Optimization of cholinesterasebased catalytic bioscavengers against organophosphorus agents, Front. Pharmacol. 9 (2018) 211, https://doi.org/10.3389/fphar.2018.00211. [8] W. Jiang, E.A. Murashko, Y.A. Dubrovskii, E.P. Podolskaya, V.N. Babakov, J. Mikler, F. Nachon, P. Masson, L.M. Schopfer, O. Lockridge, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase, Anal. Biochem. 439 (2013) 132e141, https:// doi.org/10.1016/j.ab.2013.04.018. [9] T.P. Mathews, M.D. Carter, D. Johnson, S.L. Isenberg, L.A. Graham, J.D. Thomas, R.C. Johnson, High-confidence qualitative identification of organophosphorus nerve agent adducts to human butyrylcholinesterase, Anal. Chem. 89 (2017) 1955e1964, https://doi.org/10.1021/acs.analchem.6b04441. [10] Q. Li, H. Yang, Y. Chen, H. Sun, Recent progress in the identification of selective butyrylcholinesterase inhibitors for Alzheimer's disease, Eur. J. Med. Chem. 132 (2017) 294e309, https://doi.org/10.1016/j.ejmech.2017.03.062. [11] W. Hussein, B.N. Saglik, S. Levent, B. Korkut, S. Ilgin, Y. Ozkay, Z.A. Kaplancikli, Synthesis and biological evaluation of new cholinesterase inhibitors for alzheimer's disease, Molecules (2018) 23, https://doi.org/10.3390/ molecules23082033. [12] O. Lockridge, H.W. Eckerson, B.N. La Du, Interchain disulfide bonds and subunit organization in human serum cholinesterase, J. Biol. Chem. 254 (1979)

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