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Conformational properties of the chemotherapeutic drug analogue Epothilone A: How to model a flexible protein ligand using scarcely available experimental data Jozica Dolenc, Wilfred F. van Gunsteren, Andrea E. Prota, Michel O. Steinmetz, and John H. Missimer J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00171 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Journal of Chemical Information and Modeling

Conformational Properties of the Chemotherapeutic Drug Analogue Epothilone A: How to Model a Flexible Protein Ligand Using Scarcely Available Experimental Data Jožica Dolenca,† ,*, Wilfred F. van Gunsterenb, Andrea E. Protaa, Michel O. Steinmetza,c, and John H. Missimera

a Laboratory

of Biomolecular Research, Division of Biology and Chemistry, Paul

Scherrer Institut, CH-5232 Villigen, Switzerland b Laboratory

of Physical Chemistry, Swiss Federal Institute of Technology, ETH,

CH-8093 Zurich, Switzerland c University

of Basel, Biozentrum, CH-4056 Basel, Switzerland

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ABSTRACT

Epothilones are among the most potent chemotherapeutic drugs used for the treatment of cancer. Epothilone A (EpoA), a natural product, is a macrocyclic molecule containing 34 non-hydrogen atoms and a thiazole side chain. NMR studies of EpoA in aqueous solution, unbound as well as bound to αβ-tubulin, and unbound in DMSO solution have delivered sets of NOE atom-atom distance bounds, but no structures based on NMR data are present in structural data banks. X-ray diffraction of crystals has provided structures of EpoA unbound and bound to αβ-tubulin. Since both crystal structures derived from X-ray diffraction intensities do not completely satisfy the three available sets of NOE distance bounds for EpoA, molecular dynamics (MD) simulations have been employed to obtain conformational ensembles in aqueous and in DMSO solution that are compatible with the respective NOE data. It was found that EpoA displays a larger conformational variability in DMSO than in water and the two conformational ensembles show little overlap. Yet, they both provide conformational scaffolds that are energetically accessible at physiological temperature and pressure.

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INTRODUCTION A key intracellular structure controlling cell division is the microtubule filament network. Microtubules are highly dynamic and as such are centrally involved in cell division. Cancer cells are particularly susceptible to compounds that target the αβtubulin heterodimer, the principal constituent of microtubules. Microtubule-stabilising agents such as the epothilones belong to the most potent chemotherapeutic drugs used for the treatment of cancer1. The binding of epothilones to microtubules promotes microtubule stability and, as a consequence, blocks cell entry into mitosis2. The compounds achieve this effect by binding to the taxane site of β-tubulin where they induce the structuring of the M-loop into a short helix3. Because the M-loop is a major secondary structural element establishing lateral tubulin contacts in microtubules4, this observation explains how epothilones promote microtubule stability.

Figure 1. A. Chemical structure and atom names of EpoA. B. Superimposed are the crystal structure of EpoA derived from X-ray diffraction intensities, X-ray_unbound5 (carbon backbone in grey) and the crystal structure of EpoA in complex with αβ-tubulin



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derived from X-ray diffraction intensities, X-ray_bound3 (carbon backbone in black). Yellow: sulphur; blue: nitrogen; red: oxygen. The conformational properties of unbound epothilones are poorly characterised. Availability of such information is, however, necessary to fully understand the energetics and hence the molecular mechanism of action of epothilones on tubulin and microtubules. Epothilone A (EpoA) is a cyclic molecule that can be extracted from the bacterium Sorangium cellulosum6-8. It consists of a 16-membered ring, contains 34 nonhydrogen atoms and can adopt various conformations. This has induced research on the conformational characteristics of the molecule and its derivatives8-9. Quantum-chemical methods10-11 or molecular dynamics (MD) simulations12-14 were used, but no comparison with X-ray diffraction intensities or NOE atom-atom distance bounds was reported. Using nuclear magnetic resonance (NMR) techniques and molecular dynamics (MD) simulation, the stereochemistry of the seven chiral centres of EpoA was determined8-9. The Cambridge Structural Database (CSD) contains a crystal structure of EpoA5 derived from X-ray diffraction intensities, here indicated as X-ray_unbound (Fig. 1). The structural databases do not contain structures of EpoA derived from NMR spectroscopic data, although nuclear Overhauser effect (NOE) data are available for EpoA in water15 (NOE_H2O, 18 upper distance bounds) and in DMSO9,16 (NOE_DMSO, 12 upper distance bounds) solution (Fig. 2). The EpoA-tubulin complex has been studied by electron crystallography at a low resolution of 2.9 Å17, by NMR1820,

and by X-ray crystallography at 2.3 Å resolution3. The NMR data of Ref. 18 were

used to derive a set of values for 19 torsional angles of EpoA bound to tubulin18, while those of Ref. 20 provided a set of intramolecular NOEs for EpoA bound to αβ-tubulin (NOE_bound, 20 upper distance bounds). The electron and X-ray crystallographic data were used to derive EpoA structures that showed significant differences. In addition, the unbound and bound (X-ray_bound) crystal structures derived from X-ray diffraction 4 ACS Paragon Plus Environment

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intensities showed differences (Fig. 1), in particular in the part of the macrocyclic ring from C2 till C9 and in the orientation of the thiazole side chain.

Figure 2. Graphical representation (red arrows) of the atom-atom distance bounds derived from experimental NMR NOE data for EpoA in water15 (A: NOE_H2O), in complex with αβ-tubulin20 (B: NOE_bound) and in DMSO9 (C: NOE_DMSO). The values of the distance bounds are given in Supporting Information Tables S6 and S7.

In view of the conformational uncertainty and flexibility of the EpoA molecule, MD simulations of EpoA in water and in DMSO were performed with the aim of determining the conformational space accessible to EpoA in solution while the respective NOE distance bounds derived from the NMR data are satisfied. To explore the compatibility of the characteristic structures of the MD simulations and the X-ray reflection intensities, electron densities were computed using these structures and X-ray reflection intensities. The comparison of the EpoA in water and in DMSO simulations allows for the analysis of the effect of an environment of different polarity on the conformational equilibrium of EpoA.



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METHODS Energy minimizations and molecular dynamics simulations were performed using the GROMOS bio-molecular simulation package21-23 with the force field 54A724-25.

Parametrization of Epothilone A Epothilone A (EpoA) was modelled using CHn united atoms as 34 heavy atoms and 4 explicit hydrogen atoms of which two belong to the OH groups O3-H3 and O7H7, one hydrogen atom is attached to the thiazole ring at position C19 and one hydrogen atom is associated with C17 as a part of the C16-C17 double bond (Figure 1). Treating the latter two hydrogen atoms explicitly was suggested by the results of the Automated Topology Builder (ATB)26. The ATB is a widely used on-line service for automated parameterization of small molecules. Force field parameters are derived using a multi-step procedure combining quantum-mechanical calculations with a knowledge-based approach to achieve compatibility with the GROMOS family of force fields. The ATB (revision 2014-09-22) was used to generate partial atomic charges, van der Waals interaction parameters, torsional-angle, bond-angle and bond-length parameters, see Figure S1 and Tables S1-S5. However, inspection of the ATB proposed charges and some other force-field parameters showed discrepancies with standard GROMOS values for comparable fragments or moieties. This led us to slightly modify these parameters. The torsional angles in the thiazole-bearing side chain, in particular the torsional angles C16-C17-C18-C19 and C14-C15-C16-C17, were parametrized based on a set of 6 ACS Paragon Plus Environment

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simulations in water and in DMSO in which the potential energy surface associated with the rotation of these torsional angles and the energy difference between syn and anti EpoA conformations were analyzed. In order to account for the transitions between the syn and anti conformations of the thiazole-bearing side chain an additional proper dihedral-angle type 46 with a force constant of 27 kJ mol-1, phase-shift angle of 180° and multiplicity of 2 was introduced for the C16-C17-C18-C19 torsional angle. The force-field parameters used for EpoA are reported in the Supporting Information.

Simulation Set-up The configuration of EpoA found in the crystal structure of tubulin3 provided initial coordinates for the simulations. Steepest-descent energy minimization of the starting structure in vacuum preceded its placement in a periodic cubic box filled with SPC water27. The dimensions of the box were determined by a minimum solute-wall distance of 1.0 nm and a minimum solute-solvent (non-hydrogen) atom-atom distance of 0.23 nm. These conditions yielded a cubic box of dimension 3.27 nm on a side containing 1122 water molecules. In order to relax unfavourable contacts between atoms of the solute and the solvent, a second energy minimization was performed in water while keeping the atoms of the solute harmonically, positionally restrained with force constant 25000 kJ mol−1 nm−2. The resulting configuration was submitted to an equilibration procedure in which a Maxwell distribution at 60 K provided the initial velocities. Between five periods of 20 ps each, the temperature was increased up to 300 K in increments of 60 K, while the force constants enforcing atom-positional restraints of the solute were reduced an order of magnitude in each step. During and after equilibration, solvent and solute were independently weakly coupled to a temperature bath with a coupling time of 0.1 ps28. Centre of mass motion was removed 7 ACS Paragon Plus Environment


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every 2 ps. Bond lengths of the solute and the geometry of the solvent molecules were constrained using the SHAKE algorithm29 with a relative geometric tolerance of 10-4, so the leap-frog integration time step could be set to 2 fs. For the non-bonded interactions a triple-range method with cut-off radii of 0.8 nm and 1.4 nm was used. Outside the longer cut-off radius, a reaction-field approximation30 was used with a relative dielectric permittivity of 61 for water31. Short-range van der Waals and electrostatic interactions were evaluated at every time step by using a charge-group pair-list. Longer-range van der Waals and electrostatic interactions, between pairs at a distance longer than 0.8 nm and shorter than 1.4 nm, were evaluated every fifth time step, at which point the pair-list was updated. After equilibration, the systems were also coupled to a pressure bath at 1 atm with a coupling time of 0.5 ps and an isothermal compressibility of 45.75·10-5 (kJ mol-1 nm-3)-1. Two types of molecular dynamics simulations of EpoA in water were performed. In the first water simulation, no restraints were imposed after the equilibration procedure. The resulting configurational ensemble violated two of the NOE distance bounds (H3(C3)-H6(C6) and H2@(C2)-H7(C7)) reported in ref. 15. In the second water simulation, these bounds were imposed as time-averaged atom-atom distance restraints32 for 10 ns after equilibration after which the simulation was continued for 50 ns without any restraints resulting in the configurational ensemble MD_H2O. In the 10 ns prelude to MD_H2O, the two time-averaged distance restraints32 enforcing the two above-mentioned NOE atom-atom distance bounds employed a force constant of 6000 kJ mol−1 nm−2 and a memory relaxation time of 20 ps . The simulation of EpoA in DMSO33 was set up similarly as for EpoA in water. Due to the different type of solvent molecules, the box of dimension 4.07 nm on a side 8 ACS Paragon Plus Environment

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contained 560 DMSO molecules. The isothermal compressibility used was 87.18·10-5 (kJ mol-1 nm-3)-1. The relative dielectric permittivity of DMSO used in the reaction-field approximation was 46. No distance restraints were applied in the simulation of EpoA in DMSO resulting in the configurational ensemble MD_DMSO.

Analysis of MD Trajectory Structures Trajectory coordinates and energies were stored at 2 ps intervals and used for analysis21. Least-squares translational and rotational fitting of atomic configurations and calculation of atom-positional root-mean-square differences (RMSD) between atomic configurations was based on all 38 solute atoms. The X-ray_bound structure3 and the X-ray_unbound structure5 constituted the two reference structures. Hydrogen bonds were defined by a minimum donor-H-acceptor angle of 135° and a maximum Hacceptor distance of 0.25 nm. Hydrogen bond percentages were calculated on the entire set of structures taken at 2 ps intervals. Inter-hydrogen distances in the simulations were calculated as -1/6, i.e. using r-6 averaging over the trajectory structures, where r indicates the actual hydrogenhydrogen distance (for hydrogens included in united C-atoms virtual hydrogen positions were used32) and then compared or restrained to the NOE atom-atom distance bounds as derived from NMR experiments in water15, tubulin20, and DMSO9. Pseudo-atom corrections34 were applied to the NOE atom-atom distance bounds where applicable. We considered using computation of the NOEs using a relaxation matrix approach, e.g., as specified in Ref. 35, but refrained from it in view of a lack of experimental information to compare to and to base such a calculation, comprising build-up rates, etc., on. 9 ACS Paragon Plus Environment


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A conformational cluster analysis of all 25000 recorded conformations per simulation was performed. The clustering algorithm, which uses Cartesian coordinates, has been described previously36. It uses a rotational and translational least-squares fit for every pair of configurations using all carbon atoms, then calculates the atom-positional RMSD for all atoms. Using as criterion for similarity of two structures an RMSD ≤ 0.12 nm, the number of neighbours (i.e. structures satisfying the similarity criterion) for each of the structures was determined. The structure with the highest number of neighbours was then taken as the central member of the first (most populated) cluster of structures. All the structures belonging to this cluster were then removed from the pool of structures, and the same procedure was applied to determine the second and higher clusters. The similarity criterion chosen gives a reasonable number of clusters and well populated most populated clusters for both, the MD_H2O and MD_DMSO simulations (see Supporting Information Table S8).

Rigid-body Crystallographic Refinement of EpoA Conformations The coordinates of the tubulin bound EpoA ligand (X-ray_bound) in the T2RTTL-EpoA structure (PDB ID 4I50, X-ray_apo) were either omitted (apo) or replaced by the corresponding coordinates of the superimposed unbound X-ray structure Xray_unbound (RMSD = 0.017 nm computed for 12 atoms: C1, O1, C2, O2, C9, C10, C11, C12, C13, C14, C15 and C16), of the dominant conformation MD_H2O_dom of the ensemble MD_H2O of simulated aqueous solution structures (RMSD = 0.037 nm computed for 13 atoms: C1, O1, C2, O2, C3, C4, C12, C13, C14, C15, C16, C17 and C18) or of the dominant conformation MD_DMSO_dom of the ensemble MD_DMSO of simulated DMSO solution structures (RMSD = 0.030 nm computed for 11 atoms: C1, O1, C2, O2, C12, C13, C14, C15, C16, C17 and C27) prior to three cycles of rigid body 10 ACS Paragon Plus Environment

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refinement with grouped atomic displacement parameters using the software PHENIX37 and its default input parameter settings (multi zones protocol, rigid body chains A1-438, B2-440,

C1-440,

D2-441,

E6-143,

F1-379

and

K504

for

the

ligand,

mode = *first_macro_cycle_only, target_auto_switch_resolution = 6, refine_rotation = True, refine_translation = True, max_iterations = 25, bulk_solvent_and_scale = True, euler_angle_convention = *xyz, min_number_of_reflections = 200, high_resolution = 3,

number_of_zones

=

5,

group_adp_refinement_mode

=

*one_adp_group_per_residue). The individual ligands were defined as separate chains to allow movement independent of the -tubulin chains during refinement. All the structures were refined using 129379 reflections to Rwork and Rfree values in the range of 19.1 - 19.6 and 24.5 - 24.7, respectively.

RESULTS AND DISCUSSION

Which Structure Satisfies Which Experimental Data? For the EpoA molecule, there are reports on two sets of X-ray crystallographic diffraction data, that is, intensities of X-ray reflections or squares of structure factor amplitudes, which we call X-ray_F2_bound from the crystal of the EpoA-tubulin complex3 and X-ray_F2_unbound from the EpoA crystal5. Unfortunately, the X-ray reflection intensities X-ray_F2_unbound were neither found in crystallographic data bases nor obtained from Ref. 5. There are three sets of NMR data available for which NOE intensities had been converted to atom-atom distance upper bounds. We denote NOE_H2O the bounds of the molecule in aqueous solution15, NOE_DMSO those of the molecule in DMSO solution9, and NOE_bound those of the molecule in complex with αβ-tubulin20, see Fig. 2. The three sets of distance bounds are not identical and their 11 ACS Paragon Plus Environment


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number and distribution over the molecule are insufficient to uniquely determine a conformation of EpoA. Neither the unbound (X-ray_unbound) nor the bound (X-ray_bound) structures of EpoA in crystal completely satisfy the three sets of NMR NOE atom-atom distance bounds derived for the molecule in solution (Fig. 3). The X-ray_unbound structure shows one larger (H3-H6) and two smaller (H15-H17 and H2-H7) NOE atom-atom distance bound violations for the bounds derived for EpoA in aqueous solution. Thus this X-ray crystal structure is not wholly compatible with the NMR solution data. The X-ray_bound structure also shows three atom-atom distance bound violations (H17H13, H15-H17 and H6-H23) for the bounds derived for EpoA in complex with tubulin in aqueous solution. The violations are smaller, the latter two are even insignificant.


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Figure 3. NOE atom-atom distance bound violations as function of NOE sequence number calculated for the crystal structure of unbound EpoA derived from X-ray diffraction intensities, X-ray_unbound (green bars), and the crystal structure of EpoA in complex with αβ-tubulin derived from X-ray diffraction intensities, X-ray_bound (black bars), using three sets of NOE bounds: in water (left: NOE_H2O), in complex (middle: NOE_bound) and in DMSO (right: NOE_DMSO). The NOE upper distance bounds and their violations are given in Supporting Information Table S6.



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The structural differences between X-ray_bound and X-ray_unbound lead to different NOE atom-atom distance bound violations for all three data sets NOE_H2O, NOE_bound and NOE_DMSO. This makes one wonder how well the two crystal structures of EpoA derived from X-ray diffraction intensities match the only available set of X-ray reflections X_ray_F2_bound. The electron densities 2Fobs-Fcalc generated by each of the two crystal structures while using the set of measured structure factor amplitudes X_ray_F2_bound are shown in Figure 4. Here, 𝐹𝑜𝑏𝑠 = |𝐹𝑜𝑏𝑠|𝑒 ―𝑖𝜑𝑐𝑎𝑙𝑐 and 𝐹𝑐𝑎𝑙𝑐 = |𝐹𝑐𝑎𝑙𝑐|𝑒 ―𝑖𝜑𝑐𝑎𝑙𝑐, where |𝐹𝑜𝑏𝑠| are the measured structure factor amplitudes,

|𝐹𝑐𝑎𝑙𝑐| and 𝜑𝑐𝑎𝑙𝑐 the amplitudes and phases calculated from a structure in the crystal unit cell. The upper panels of Figure 4 show, as expected, that the structure X-ray_bound

fits

the

density

calculated

using

the

measured

reflections

X-ray_F2_bound, while the lower panels indicate that this is not the case for the structure X-ray_unbound.

Figure 4. Electron densities, 2Fobs - Fcalc of EpoA calculated using the structures X-ray_apo, X-ray_bound and X-ray_unbound relative to the measured X-ray reflection intensities X-ray_F2_bound. (A) Electron density omit map after rigid-body refinement 14 ACS Paragon Plus Environment

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of the T2R-TTL-apo structure. The EpoA molecule has been superimposed onto the density after refinement to highlight the agreement with the bound conformation. (B) Electron density map after rigid-body refinement of the bound crystal structure derived from X-ray diffraction intensities X-ray_bound. (C) and (D) Electron density maps after rigid-body refinement of the unbound crystal structure derived from X-ray diffraction intensities X-ray_unbound in two different orientations. The σA‐weighted 2mFobs ‐ DFcalc (blue mesh) and mFobs ‐ DFcalc (green and red mesh) omit maps38 are contoured at + 1.0σ and +/- 3.0σ, respectively. "m" is a figure of merit relating to the phase information of a reflection; the contributions of reflections with large phase errors are weighed less. "D" is a weighing factor incorporating an overall estimate of atomic coordinate errors. The effect of these factors is the reduction of noise in the map. Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.8.4.2 Schrödinger, LLC).

From this analysis it is apparent that the different structures of EpoA derived from experimental data do not completely satisfy these. Therefore, we set out to derive a set of structures of EpoA that satisfies the NMR NOE bounds as derived from measurements of EpoA in aqueous solution and investigate whether these structures are compatible with the measured X-ray reflections for the crystal of bound EpoA and with the NMR NOE bounds for EpoA in complex and in DMSO.

Derivation of Structures of EpoA Compatible with Measured NMR Data The standard way to derive molecular structure from measured values of observable quantities is to perform molecular dynamics (MD) simulations using a restraining term added to the potential energy function which imposes an energy penalty upon structures that deviate from the measured values of a particular quantity, in the present case the NOE atom-atom distance bounds32,

39.

Since the number of

NOE atom-atom distance restraints available is relatively low: 18 in water, 20 in



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complex, and 12 in DMSO, compared to the number of internal degrees of freedom of the 34 non-hydrogen atoms of EpoA (3x34-6=96) or comparable to the number of rotatable torsional angles in EpoA (18), the set of low energy structures resulting from the MD structure refinement will depend critically on the quality of the force field used in the structure determination40. In the present case one of the most recent GROMOS bio-molecular force-field parameter sets, 54A724-25, was used.

Comparison of the NMR Compatible Solution Structures of EpoA with the X-ray Crystal Data Figure 5 shows that the backbone atom-positional root-mean-square differences (RMSD) of the MD trajectory structures from the bound and unbound X-ray structures fluctuate between 0.1 to 0.3 nm. The solution structures stay on average slightly closer to the X-ray_unbound structure than to the X-ray_bound structure.

Figure 5. Atom-positional root-mean-square differences (RMSD) for the EpoA backbone atoms between the crystal structures derived from X-ray diffraction intensities


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X-ray_bound (top panels) and X-ray_unbound (bottom panels) for the simulations MD_H2O (left-hand panels) and MD_DMSO (right-hand panels). A conformational cluster analysis of the trajectories of MD_H2O and MD_DMSO, each augmented with either the X-ray_bound structure or the Xray_unbound structure, shows that the latter structure is present in neither MD_H2O nor MD_DMSO, whereas the X-ray_bound structure occurs in both solutions, 1.3 % in MD_H2O and 2.6 % in MD_DMSO (see Supporting Information Table S8). While the low occurrences of the X-ray_bound structure imply a conformational selection mechanism upon binding, the absence of X-ray_unbound structure in the simulated ensembles may be due to crystal contacts (intermolecular O12…H3-O3 and O5…O7H7 hydrogen bonds between neighbouring EpoA molecules in different asymmetric units of the crystal and a hydrogen bond from N18 of the EpoA thiazole ring to the O-H group of the methanol molecule present in the crystal) that influence the conformation of the X-ray_unbound structure of EpoA. As expected, both sets of MD trajectory structures satisfy the three sets of NOE atom-atom distance bounds better than the two X-ray crystal structures (compare Figures 6 and 3).



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Figure 6. NOE atom-atom distance bound violations as function of NOE sequence number calculated for the MD trajectory structures of the simulations MD_H2O (blue bars) and MD_DMSO (red bars) using three sets of NOE bounds: in water (left: NOE_H2O), in complex (middle: NOE_bound) and in DMSO (right: NOE_DMSO). The NOE upper distance bounds and their violations are given in Supporting Information Table S7.

An electron density calculation for the dominant conformation MD_H2O_dom of the ensemble MD_H2O inserted into the crystal unit cell at the position of the EpoA molecule shows that this dominant aqueous solution structure yields only partially an 18 ACS Paragon Plus Environment

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appropriate electron density in the crystal (upper panels of Figure 7). The same observation holds for the dominant conformation MD_DMSO_dom of the ensemble MD_DMSO inserted into the crystal unit cell (lower panels of Figure 7). Thus in aqueous as well as in DMSO solution the dominant conformation does not match the X-ray crystallographic data X-ray_F2_bound as well as the X-ray_bound structure.

Figure 7. Electron densities, 2Fobs - Fcalc of parts of EpoA calculated using the dominant conformations MD_H2O_dom and MD_DMSO_dom (central members of the most populated conformational clusters) of the ensembles MD_H2O and MD_DMSO inserted into the crystal unit cell at the position of EpoA and the measured X-ray reflection intensities X-ray_F2_bound. (A) and (B) Electron density maps after rigid body refinement of the MD_H2O_dom structure in two different orientations. (C) and (D) Electron density maps after rigid body refinement of the MD_DMSO_dom structure in two different orientations. The σA‐weighted 2mFobs-DFcalc (blue mesh) and mFobs ‐DFcalc (green and red mesh) omit maps38 are contoured at + 1.0σ and +/- 3.0σ, respectively. "m" is a figure of merit relating to the phase information of a reflection; the contributions of reflections with large phase errors are weighed less. "D" is a weighing factor incorporating an overall estimate of atomic coordinate errors. The effect of these factors is the reduction of noise in the map. Figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 1.8.4.2 Schrödinger, LLC). 19 ACS Paragon Plus Environment


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Analysis of the Simulated and Experimentally Derived EpoA Structures Due to its many rotatable bonds EpoA can adopt a variety of different conformations. To characterize the conformational variability of EpoA and the differences between its unbound conformations in water and in DMSO and its tubulinbound conformation the torsional angles in the macrocycle and in the thiazole side chain were analysed and compared.


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Table 1. Torsional-angle values that differ more than 30° among any of the X-ray crystal structures and the three most dominant structures of the ensemble MD_H2O. Grey denotes the torsional-angle values of the dominant structures deviating by substantially more than 30° from the values of the crystal structure X-ray_unbound in the second column. Multiple entries are given for those angles that fluctuate significantly; they are in order of decreasing frequency. The last two columns contain data for a NMR structure of tubulin-bound EpoA in aqueous solution and a crystal structure of EpoA in methyl tert-butyl ether as quoted in Ref. 18.

torsional angle

X-ray

MD_H2O

bound

unbound

C15-O1-C1-C2

172

174

O1-C1-C2-C3

-36

C1-C2-C3-C4

cluster 1

NMR18

cluster 2

cluster 3

177

185

83

176.3±1.3

174.2

146

-79

91, -39

98

-124.3±1.2

156.7

-167

162

175

137

-173

-152.5±0.2

165.4

C2-C3-C4-C5

-59

72

-60

-62

-63

-51.7±0.1

73.0

C5-C6-C7-C8

-60

-63

-151

-142

-144

-70.0±0.8

-64.0

C6-C7-C8-C9

-68

-80

167, 77

65, 143

153, 55

-74.8±0.3

-79.2

C7-C8-C9-C10

169

159

-51, 75, 155

83

120,-45

164.1±0.8

159.0

C8-C9-C10-C11

-152

176

-143

-178

-160

-171.9±0.4

176.8

C9-C10-C11-C12

-170

174

-80, 180, 85

-179

149

-178.0±0.4

174.8

C13-C14-C15-C16

153

160

169

89, 145

74

n.a.

n.a.

C13-C14-C15-O1

-88

-78

-65

-147, -91

-158

-62.6±1.0

-82.6

C14-C15-O1-C1

169

168

158

89, 157

140

179.5±0.5

159.6

C14-C15-C16-C17

-147

84

-113

-118

-119

-129.7±1.3

-118.5


bound

X-ray18 unbound

Journal of Chemical Information and Modeling 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

Page 22 of 38

Table 2. Torsional-angle values that differ more than 30° among any of the X-ray crystal structures and the four most dominant structures of the ensemble MD_DMSO. Grey denotes the torsional-angle values of the dominant structures deviating by substantially more than 30° from the values of the crystal structure X-ray_unbound in the second column. Multiple entries are given for those angles that fluctuate significantly; they are in order of decreasing frequency. The last two columns contain data for a NMR structure of tubulin-bound EpoA in aqueous solution and a crystal structure of EpoA in methyl tert-butyl ether as quoted in Ref. 18.

torsional angle

X-ray

MD_DMSO cluster 1

cluster 2

cluster 3

cluster 4

NMR18

X-ray18

bound

unbound

bound

unbound

C16-C17-C18-N18

-178

-165

0

0

7, -9

179

180.0±0.3

-7.6

O1-C1-C2-C3

-36

146

-89

75,-43

70

73,-39

-124.3±1.2

156.7

C1-C2-C3-C4

-167

162

157, 180

-177,147,83

76

147,-179

-152.5±0.2

165.4

C3-C4-C5-O5

98

111

80,151,-35

85, -39

-32

85

n.a.

n.a.

C3-C4-C5-C6

-83

-75

-109, 151

-101,145

152

-100

-43.0±1.8

-75.9

C5-C6-C7-C8

-60

-63

73,-71

-51,83

88

-54

-70.0±0.8

-64.0

C8-C9-C10-C11

-152

176

172

-169

-157,-61

-164

-171.9±0.4

176.8

C9-C10-C11-C12

-170

174

77, 163

175

169, 127

175

-178.0±0.4

174.8

C13-C14-C15-C16

153

160

-177

85,145

74

89,135

n.a.

n.a.

C13-C14-C15-O1

-88

-78

-51

-145,-91

-159

-141,-101

-62.6±1.0

-82.6

C14-C15-O1-C1

169

168

151

85,157

124

87,163

179.5±0.5

159.6


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In Table 1 and Table 2 the values for a set of torsional angles of EpoA for which the two crystal structures and the MD generated aqueous and DMSO solution structures show differences are listed. Not surprisingly, the largest differences between the EpoA conformations in different environments occur in the macrolide part. About eight torsional angles show sizeable fluctuations. Their distributions are shown in Figure 8. They indicate sizeable flexibility in three parts: O1-C14, C7-C11 and C1-C3 of the 16membered ring affecting the positioning of the biologically relevant O3-H3 group. Tables 1 and 2 also present the torsional-angle values found by repeated single-structure refinement18 based on NMR data on EpoA bound to tubulin and X-ray crystal data on unbound EpoA. The uncertainty in individual torsional-angle values, between 0.1° and 1.8°, of bound EpoA seems quite small. This may be due to either the limited conformational freedom allowed in the binding site or to the single-structure procedure, in contrast to a time-averaging procedure32, to obtain structures compatible with experimentally

observed

data.

Single-structure

refinement

can

lead

to

the

underestimation of the structural variability within a molecule41-42. This applies to X-ray crystallographic single-structure refinement43-45 as well as to that based on NMR46. In DMSO EpoA adopts conformations that differ substantially from the conformations found in water or in the binding pocket of β-tubulin. Significant differences are observed along almost the whole macrolide ring as well as in the thiazole side chain, where the torsional angle C16-C17-C18-N18 undergoes a rotation around the C17-C18 bond leading to a change in the orientation of the thiazole side chain. The thiazole side-chain rotational fluctuations observed in DMSO solution are in agreement with the experimental results obtained by NMR spectroscopy in DMSO/water mixtures.



Page 24 of 38

Figure 8. Distributions of torsional angles in the MD simulation of EpoA in water (blue) and in DMSO (red). 25000 trajectory structures were analysed.

In Figure 9 the most populated conformational clusters of unbound EpoA in water and DMSO solutions are shown and compared with the X-ray_unbound and X-ray_bound EpoA structures. The ensemble of MD generated structures in water is represented by the central members of its three most populated conformational clusters, containing 94%, 5% and 0.2% of the 50 ns trajectory structures, and the ensemble of MD generated structures in DMSO is represented by the central members of its four most populated conformational clusters, containing 69%, 14%, 6% and 4% of the 50 ns trajectory structures. Figure 10 shows that there is little overlap between the EpoA conformations in water and in DMSO.


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Figure 9. Upper panels: Superposition of the most dominant conformations, i.e. central members of the most populated conformational clusters, derived using a conformational cluster analysis of the MD trajectory structures of EpoA from MD_H2O (A) and MD_DMSO (B) simulations. Lower panels: Superposition of the X-ray_unbound and X-ray_bound EpoA structures with the central member of the most populated cluster from MD_H2O (C) and MD_DMSO (D) simulations. A backbone RMSD similarity criterion of 0.12 nm was used. Red: first cluster; green: second cluster; blue: third cluster; grey: fourth cluster; yellow: X-ray_unbound EpoA structure; brown: Xray_bound EpoA structure. Molecules were superimposed using backbone carbon atoms.



Page 26 of 38

Figure 10. Combined conformational cluster analysis of the 50 ns trajectories MD_H2O (blue) and MD_DMSO (red) showing the contribution of each of the two trajectories to the clusters.

Comparison of the atom-positional root-mean-square fluctuations (RMSF) for EpoA in aqueous and DMSO solutions (Figure 11) reveals enhanced flexibility of EpoA in the DMSO environment compared to the aqueous environment. The X-ray single-structure crystallographic refinements leading to the structures X-ray_bound and X-ray_unbound also generated isotropic atomic B-factors. The Bfactor Bi of atom i can be converted40 to an atom-positional root-mean-square fluctuation (RMSF) value RMSFi for atom i using the formula RMSFi = (3Bi/(8π2))½. One should note, however, that the quantities Bi and RMSFi are differently defined quantities40. The crystallographic B-factor Bi is derived from X-ray diffraction intensities by fitting the radius of the electron density at position i to match the corresponding electron density profile, and the mean-square atom-positional fluctuation MSFi of atom i is obtained from a simulation. Bi constitutes an average for a specific position i over different atoms passing through this position, while MSFi is an average 26 ACS Paragon Plus Environment

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for a specific atom i over its trajectory of positions. This means that the abovementioned relation will only hold for small amplitude atomic motion. Bi values should by definition not become larger than the size of an atomic position, while RMSFi values can take any value as long as an atom moves sufficiently. In a crystal, the atomic motion may be restricted, while in solution this will be less so. These considerations explain the differences between the simulated and crystallographically refined values in Figure 11.

Figure 11. Atom-positional root-mean-square fluctuations (RMSF) of non-hydrogen atoms of EpoA in the simulations MD_H2O (blue) and MD_DMSO (red) averaged over 50 ns of simulation time. The atomic B-factors resulting from single-structure crystallographic refinement of EpoA, converted to RMSF values, are shown in green (X-ray_bound) and magenta (X-ray_unbound).

We note that despite the presence of hydrogen bond donors and acceptors in the EpoA molecule no intra-molecular hydrogen bonds were observed in any of the simulations. The only exception is a water mediated hydrogen bond between N18 and 27 ACS Paragon Plus Environment


Page 28 of 38

O2 that was observed in the MD_H2O simulation. The hydroxyl and carbonyl groups on the macrocycle are thus available for inter-molecular interactions with the solvent or within the binding pocket. Analysis of the solute-solvent interactions showed a smaller number of solute-solvent hydrogen bonds in DMSO than in water, which is due to the difference in size of these solvent molecules. In DMSO only 2 hydrogen bonds are present for more than 5% of the simulation time (involving solute atoms H3 and H7), whereas in water there are 9 (involving solute atoms N18, O12, O7, H7, O5, O3, H3, O2, O1).


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CONCLUSIONS The conformational properties of EpoA were investigated using MD simulation in view of measured data including X-ray crystallographic diffraction intensities for EpoA bound to αβ-tubulin and three sets of NOE atom-atom distance bounds derived from NMR spectroscopic data: for EpoA in aqueous and DMSO solution, and for EpoA bound to αβ-tubulin in aqueous solution. Neither crystal structure derived from X-ray diffraction intensities wholly satisfies the different sets of NOE atom-atom distance bounds. The latter are insufficient in number and distribution over the molecule to uniquely determine a dominant EpoA conformation. The three NOE data sets are not identical, reflecting the effect of the different environments on the conformational ensemble of EpoA. The MD simulated trajectories, Boltzmann-weighted conformational ensembles, match the NOE data sets slightly better than the crystal structures derived from X-ray diffraction intensities. In the ensemble representing EpoA in water, the bound structure, X-ray_bound, as found by X-ray crystallography, is present, which means, it is energetically accessible at room temperature and pressure in aqueous solution. Surprisingly, no conformations close to the X-ray_unbound structure are found in this ensemble. This structure may be influenced by crystal contacts that perturb the conformational ensemble in solution. The conformations dominant in solution do not match the electron density of bound EpoA. The hydrogen-bond donors and acceptors of EpoA do not form intra-molecular hydrogen bonds. The MD generated structural ensembles in water and in DMSO display little conformational overlap. The distributions of values of torsional angles along the macrocyclic ring show significant differences. Although both solvents, water and DMSO, are polar, DMSO allows EpoA more conformational variability than water. The 29 ACS Paragon Plus Environment


Page 30 of 38

MD simulations of EpoA in the different solvents show different conformational equilibria, which means that its conformation may change with a changing environment. Thus the dominant structure in a cell will depend on the composition of the solvent in a cell. The different dominant conformers of EpoA in water and in DMSO may serve as a basis for the design of smaller, less complex EpoA-like molecules that may serve as tubulin-targeting chemotherapeutic drug.


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ASSOCIATED CONTENT Supporting Information. EpoA atomic numbering scheme used in GROMOS 54A7 force-field parameter tables (Figure S1); GROMOS atom sequence numbers and names, integer atom codes, mass atom type codes and atom charges and ATB charges for EpoA (Table S1); GROMOS and ATB bond types for EpoA (Table S2); GROMOS and ATB bond-angle types for EpoA (Table S3); GROMOS and ATB improper dihedral-angle types for EpoA (Table S4); GROMOS and ATB proper dihedral-angle types for EpoA (Table S5); hydrogen atom pairs with NOE upper distance bounds and their violations for the EpoA crystal structures X-ray_unbound and X-ray_bound using NOE bounds in water, in complex with αβ-tubulin and in DMSO (Table S6); hydrogen atom pairs with NOE upper distance bounds derived from NMR experiments for EpoA in water, in complex with αβ-tubulin and in DMSO, and their violations for MD trajectories MD_H2O and MD_DMSO (Table S7); conformational clustering analysis of MD_H2O and MD_DMSO trajectories showing the sensitivity of the clustering results on the RMSD cut-off value (Table S8).



Page 32 of 38

AUTHOR INFORMATION Corresponding Author * Jožica Dolenc E-mail: [email protected] ORCID: 0000-0001-5892-2700

Present Addresses † Chemistry | Biology | Pharmacy Information Center, ETH, CH-8093 Zurich, Switzerland Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by a grant from the Swiss National Science Foundation (31003A 166608 to M.O.S.). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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This work was supported by a grant from the Swiss National Science Foundation (31003A 166608 to M.O.S.). We thank Prof. Angeles Canales for providing the experimental NOE upper distance bounds for EpoA bound to tubulin.

ABBREVIATIONS DMSO, dimethyl sulfoxide; MD, molecular dynamics; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; RMSD, root-mean-square difference; RMSF, root-mean-square fluctuation; SPC, simple point charge; EpoA, Epothilone A; GROMOS, Groningen Molecular Simulation software; CSD, Cambridge Structural Database.

PDB ID CODES USED 4I50



Page 34 of 38

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For Table of Contents Use Only

Conformational Properties of the Chemotherapeutic Drug Analogue Epothilone A: How to Model a Flexible Protein Ligand Using Scarcely Available Experimental Data Jožica Dolenc*, Wilfred F. van Gunsteren, Andrea E. Prota, Michel O. Steinmetz, and John H. Missimer


Conformational Properties of the ... - ACS Publications

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