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Exploring the Active Centre of LSD1/CoREST Complex by Molecular Dynamics Simulation Utilizing its Co-Crystallized Cofactor Tetrahydrofolate as a Probe Waleed A. A Zalloum, and Hiba M Zalloum J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00256 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Journal of Chemical Information and Modeling is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Chemical Information and Modeling

Exploring the Active Centre of LSD1/CoREST Complex by Molecular Dynamics Simulation Utilizing its Co-Crystallized Cofactor Tetrahydrofolate as a Probe

Waleed A. Zalloum1*, Hiba M. Zalloum2* 1

Department of pharmacy, Faculty of health science, American University of Madaba, P.O Box

2882, Amman 11821, Jordan 2

Hamdi Mango Research Centre for Scientific Research, The University of Jordan, Amman

11942, Jordan *Corresponding Authors Waleed A. Zalloum [email protected], [email protected] Hiba M. Zalloum [email protected], [email protected]

Author Contributions W.A. Z and H.M Z. contributed equally

ACS Paragon Plus Environment

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Abstract Epigenetic targeting of cancer is a recent era to manipulate the gene without destroying the genetic material. Lysine-specific demethylase 1 (LSD1) is one of the enzymes associated with the chromatin for post-translational modifications, where it demethylates lysine amino acid in the chromatin H3 tail. Many studies showed that inhibiting LSD1 could potentially be used to treat cancer epigenetically. LSD1 is associated with its corepressor protein CoREST, and uses tetrahydrofolate as a cofactor to accept CH2 from the demethylation process. In this study the cocrystallized cofactor tetrahydrofolate was utilised to find possible binding regions in the active centre of the LSD1/CoREST complex. Also, the flexibility of the complex has been investigated by molecular dynamics simulation and subsequent analysis by clustering and principal component analysis. This research supported other studies and showed that LSD1/CoREST complex exists in two main conformational structures, the open and closed. Furthermore, this study showed that tetrahydrofolate stably binds to the LSD1/CoREST complex, in its open conformation, at its entrance. Then, it binds to the core of the complex inducing the closed conformation. Furthermore, the interactions of tetrahydrofolate to these two binding regions and the corresponding binding mode of tetrahydrofolate were investigated to be used in structure based drug design.


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Introduction Cancer is well known as a worldwide public health problem needs a huge effort for treatment. 1-3 There is a huge research on the treatment of cancer through screening of organic compounds alongside the clinically available chemotherapeutic agents.4-8 However, these chemotherapeutic agents are not specific in their action and mostly associated with high toxicity.9,10 Accordingly, it is far too important to understand different pathophysiological mechanisms underlying cancer initiation and progression at molecular level, and finding specifically related intracellular drug targets which could revolutionise the finding of future novel potent and selective cancer treatment.11-17 Epigenetics has emerged as the study of modifications of gene expression without altering the genetic material.11,12,18,19 Gene expression is controlled by post-translational modifications of its associated histones.20,21,11 These modifications play a critical role in gene silencing and they are involved in the disease pathology. The epigenetic control could be targeted to control expression of certain gene without destroying the gene itself.21,11 One of the epigenetic post-translational histones modifications is Lysine methylation, which had been considered as permanent modification.18,22 Recently, it has been found that flavin adenine dinucleotide (FAD) dependent amine oxidase enzyme, called lysine-specific demethylase 1 (LSD1), demethylates histone mono or dimethylated lysine by redox reaction.22 LSD1 is associated with other transcriptional factors such as its co-repressor CoREST to regulate variety of genes including the expression of tumour suppressor gene. It has been reported in different research studies that LSD1/CoREST complex has a role in cancer disease, where it is overexpressed in various cancer cells such as breast, prostate and gastric cancers.22,23 Furthermore, inhibition of LSD1/CoREST complex showed increased methylation at the histone H3 lysine 4 (H3K4), which reactivates the expression of tumour suppressor genes in different types of cancer.22-24 Accordingly, LSD1/CoREST complex is considered as an important intracellular epigenetic target for the development of new anticancer drugs by reactivating the silenced tumour suppressor gene without destroying the gene itself. This would target cancerous rather than normal cells, which potentially enables selectively targeting cancer.11,22-24



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Due to the importance of LSD1/CoREST complex in cancer pathogenesis, different research groups have studied its structure and dynamics in its substrate-bound and unbound forms.25-27 Some studies showed that LSD1/CoREST complex has an open-closed nanoscale clamp that is induced by its substrate H3 histone tail and used for the molecular recognition.25 Furthermore, other research groups studied the bases of molecular recognition of the substrate in order to design novel inhibitors based on the identifying key amino acids and the catalytic mechanism of lysine demethylation.27 The druggability of the complex has been studied by different computational algorithms to expand the possibility of designing LSD1/CoREST complex inhibitors.28 LSD1/CoREST complex uses tetrahydrofolate (THF) as a cofactor, where it accepts the methyl group resulted from the demethylation process of the methylated lysine residue.29,30 This produces

5,10-methylene-tetrahydrofolate

(5,10-CH2-tetrahydrofolate),

biochemical pathway is converted to THF again.

29

which

by

other

The function of tetrahydrofolate as methyl

group scavenger in an enzymatic reaction necessitates its close proximity to the reaction centre as well as its functional groups are well-oriented for proper functioning as a cofactor involved in the enzymatic reaction.31,32 Several

LSD1/CoREST

inhibitors

were

reported

to

have

anticancer

properties.33-37

LSD1/CoREST was inhibited by MAOs inhibitors, due to its similarity to monoamine oxidases (MAOs), such as tranylcypromine (TCP) and pargyline scaffolds.34 Derivatives of these scaffolds show activity against MAO A or MAO B, which limited their preclinical studies as LSD1/CoREST inhibitors due to their low selectivity.34 Also, there are few peptide derivatives of pargyline that showed inhibitory effect on LSD1.37 Most of these inhibitors are irreversible binders which denature the enzyme. Furthermore, the peptide derivatives of these scaffolds have limitation in their delivery to the nucleus.38 Generally, the available LSD1/CoREST inhibitors showed poor selectivity, low potency, or in vivo toxicity, which limit further their development to anticancer drugs.35 Accordingly, this research focuses on probing possible binding regions in the active centre of LSD1/CoREST complex utilising its cofactor tetrahydrofolate using molecular dynamics simulation (MD), and exploring the presence of different conformational ensembles of the complex. Moreover, this study investigates the binding modes of THF, due to its optimal biding to its binding site, in the active centre of LSD1/CoREST complex for future


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structure-based design, such as molecular docking. Results of this study will be used to design novel potent, selective, low toxicity and reversible LSD1/CoREST complex inhibitors as anticancer agents. Materials and Methods Molecular dynamics simulations The initial X-ray structure of LSD1/CoREST-THF complex was downloaded from protein databank (PDB code 4KUM with a resolution of 3.05 Å).30 Atomic point charges for THF and FAD were derived from AM1-BCC charge model with ANTECHAMBER in AMBER14.39 The AMBER14 coordinate and topology files were prepared using ff14sb force field for protein and GAFF force field for THF and FAD. The MD simulation was run using an explicit water TIP3PBOX octahedral solvent box model with 8Å cut, and no ions were needed for neutralization. Water was first minimised for 10000 cycles using steepest descent and then conjugate gradient algorithms. Then, the whole system was minimised for 2500 cycles using steepest descent followed by conjugate gradient algorithms. Water was equilibrated for 40 ps at constant volume with periodic boundaries, where restraints on LSD1/COREST-THF complex were hold with weak strength through the equilibrium stage, a force constant of 10 Å as position restraint. Then, the whole system was equilibrated for 200 ps using constant pressure periodic boundaries with no restraints. This was followed by unrestrained molecular dynamics simulation at constant temperature of 300 K and constant pressure of 1 atm for 1µs. Cutoff distance for the calculation of non-bonded forces was set to 10 Å. HMassRepartition was used to shift the mass of all hydrogen atoms of LSD1/CoREST-THF complex to 3.024 Da. The masses of attached heavy atoms were considered by adjusting their masses so the total mass of each pair is unchanged. This allowed us to use an integration time step of 4 fs during MD.34 The energies and atomic coordinates were saved to the trajectory file every 4 ps. SHAKE bond length constraint involving hydrogen atoms was turned on. Clustering and Principal Component Analysis The first 50 ns of the MD simulation were removed from the analysis, and the remaining trajectories were imaged and then water molecules were stripped off. The frames used for the subsequent studies were selected every second frame. All trajectories were aligned against the



first frame of the MD using the protein residues only. Then they were clustered by DBSCAN algorithm41,42 implemented in cpptraj of AMBER14 on THF cofactor using minimum points of 4 and epsilon of 2.5 with no frame orientation (no fit), since they previously are rms against first frame. This clustered all frames according to the THF cofactor to explore the binding regions of LSD1/CoREST complex to THF cofactor. Principal component analysis (PCA) is used to reduce dimensionality and find patterns in a highdimensional data, such as MD trajectories, in a way that explains the data variance.43,44 Application of PCA on trajectory frames describes major conformational changes during the time of the MD simulation.45 Accordingly, PCA has been used in this project to reveal the concerted motions of LSD1/CoREST-THF complex MD trajectories, which would indicate major binding regions for THF inside the LSD1/CoREST complex active centre. PCA was run on Cartesian coordinates which has prove to be valuable method to study conformational changes.45 The covariance matrix was calculated using the imaged trajectories based on protein residues and THF using heavy atoms only. PCA is based on the diagonalization of the covariance matrix of atomic positional fluctuations, which yields a set of eigenvectors and eigenvalues. The first one or two eigenvectors with highest variance in data are important for describing the significant motions of protein and the cofactor.45 There are some criteria used to determine the number of components that could be considered.46,47 The first guideline is to plot the size of each eigenvalue and find the point where the graph goes from the steep to flat, this point is called elbow and the plot is called the scree plot. Then keep the components before this elbow for consideration. The second guideline is to keep eigenvalues that are larger than 1, this is called Kaiser criterion.46,47 In this study, we used the first method to decide the number of eigenvectors included in the analysis. Results Stability of LSD1/CoREST Complex during the MD Simulation The molecular dynamics of LSD1/CoREST-THF complex in solution has been investigated for 1µs using one x-ray structure that contains THF and FAD bound to LSD1/CoREST complex. Figure 1A shows the rmsd of the backbone Cα atoms, which only was calculated for the amino acid residues of LSD1/CoREST complex, during the MD simulation in reference to the first


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frame of MD. It is clear from the figure that CoREST chain is stable during the MD simulation, as inferred from the stable rmsd fluctuation (black). The rmsd has increased to 4.0 Å during the first 1.5 ns of the MD simulation and continued fluctuating around this average for the rest of the MD trajectory. Some frames are highly deviated (up to 6 Å), which could be due to the flexibility at termini.On the contrary, LSD1 chain has highly deviated from the initial frame of the MD simulation according to rmsd fluctuation in figure 1A (red), where it fluctuated between 2.5 and 9 Å. The pattern of rmsd fluctuation for LSD1 chain shows that there could be ensembles of frames during the MD simulation with different structures, which could indicate the presence of other conformational structures of this chain. Root mean square fluctuation (RMSF) for each amino acid in LSD1/CoREST complex was calculated to find amino acid residues of high fluctuation (Figure 1B). It is clear from the figure that most of CoREST chain amino acids have low RMSF, around 2 Å. However, amino acids 667 to 674 (N-terminal) and 799 (C-terminal) have higher RMSF values (up to 7 Å), which are predicted to be flexible because of their terminal position (Figure 1C). The stable RMSF of most CoREST residues is in accordance with the rmsd fluctuation in figure 1, which indicates the stability of this chain during the long MD simulation. Figure 1B also shows that LSD1 chain has deviated from the starting frame for a wider range of amino acids compared to CoREST chain. Amino acids 1 to 3, 33 to 35, 66, 69 to 74, 105, 106, 232, 280, 283 to 323, 343 to 352, 619 and 664 to 666 have high RMSF values (up to 10 Å). Amino acids 1 to 3 (N-terminal) and 664 to 666 (C-terminal) are predicted to be flexible due to their terminal position (Figure 1C). It is inferred from figure 1C that the rest of highly fluctuating amino acids in LSD1 chain are not terminal, which could indicate the presence of other conformational ensembles. Most of the highly fluctuating amino acids in the core of the complex are exposed to the solvent, which would contribute to the flexibility of these residues. Amino acids ILE285, ALA295, PHE308, VAL310, SER312, LEU317 and CYS321 are buried and have high RMSF values, which would exclude the contribution of the solvent to the flexibility of these residues.



A

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B N-terminal LSD1

N-terminal CoREST

C

Figure 1. A) rmsd of the backbone Cα atoms for LSD1 (red) and CoREST (black) amino acids in reference to the first frame of the 1µs MD simulation. The rmsd values were calculated for LSD1 and CoREST separately. B) RMSF of the backbone Cα atoms for residues of LSD1 (red) and CoREST (black) during the 1µs MD simulation. The RMSF values were calculated for LSD1 and CoREST separately. C) FASTA sequence of LSD1/CoREST complex with the corresponding secondary structure elements α-helix (orange) and β-sheet (blue). LSD1 and CoREST chains are coloured red and black respectively, while amino acids with RMSF more than 4 Å have been annotated by bold font. This figure has been produced by Discovery Studio 3.0 visualizer.42

PCA and Clustering of LSD1/CoREST Complex


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Principal component analysis was carried out to investigate the concerted motion of LSD1/CoREST complex in order to identify the presence of any conformational ensembles according to results shown in figure 1.45 The conformational changes of LSD1/CoREST complex could be due to the binding of tetrahydrofolate to its active site as inferred from the rmsd plot in figure 1.49 Also, PCA combined with trajectory clustering could explore new binding sites in the active centre of LSD1/CoREST complex.39,44 Principal components (PC) are calculated from the Cα backbone atoms coordinate covariance matrix of the MD simulation by projection of eigenvectors, which could investigate the dominant protein motion.50 First 100 principal components were calculated for LSD1/CoREST complex MD trajectory, where the contribution of first PC to the complex motion is the most significant which contributes by 73.6%. The second and third PCs contribute by 9.5% and 3.8% respectively. According to these results, LSD1/CoREST complex could present in different conformational ensembles, where the first conformational ensemble structure is the most dominant. Clustering of the 1µs MD simulation was performed in order to find 3D structures of each conformational ensemble according to PCA results, which will be employed for future molecular docking studies. For this purpose, the long MD trajectory was clustered using dbscan clustering algorithm for all heavy atoms in LSD1/CoREST complex amino acids. The complex was clustered based on THF cofactor after aligning the MD trajectory to the first frame using amino acids of the complex. The alignment of the complex using amino acids created a common reference where the clustering algorithm, which was based on THF, can detect the rmsd differences, which generated the binding position of THF cofactor. Figure 2 shows the projection of the first three PCs sorted by the first three clusters of the dbscan algorithm. The MD trajectory has clustered into 16 clusters with first and second clusters are the most dominant. It is obvious from figure 2 that the first three clusters are well separated indicating different conformational space for each cluster. Since the first and second clusters are the most dominant, we will precede the analysis using representative structures from these clusters.



Figure 2. Projection of PC1, PC2 and PC3 sorted by first (magenta), second (green) and third (cyan) clusters.

Representative structures from first and second clusters have been superimposed (each chain separately) against the x-ray structure 4KUM (Figure 3). Residues with high RMSF values during the MD simulation are coloured red in LSD1 chain and yellow in CoREST chain and represented in figure 3A. Superposition of the CoREST chain (figure 3C) shows that the core of this chain is stable during the long MD simulation, however it has high fluctuation at termini (yellow colour in figure 3A). On the contrary, LSD1 chain fluctuated during the MD simulation (red colour in figure 3A), which indicate the presence of different conformational ensembles or flexibility of the chain. According to clustering results, LSD1 chain exists in two different structures as shown in figure 3B. Inspection the superimposed LSD1 structures in figure 3B shows that first conformational cluster structure is close to the x-ray structure, whereas the second conformational cluster structure is highly deviated from the x-ray structure at the lower part of the tower.


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A

B

C

Tower

AOD

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 3. A) 3D structure of LSD1/CoREST complex (4KUM) with the highly deviated residues coloured red in LSD1 chain and yellow in CoREST chain. The tower and the amino oxidase domains (AOD) are represented. B) LSD1 and C) CoREST chains superposition for 4KUM (black), a representative structure of the first cluster (blue) and a representative structure of the second cluster (green).

Binding of THF to the active centre of LSD1/CoREST complex The binding of THF to the active centre of LSD1/CoREST complex was investigated to probe possible binding regions that are emerged due to its binding. As mentioned above, the 1µs MD simulation resulted in two populated clusters based on the rmsd of all heavy atoms of protein residues only without fitting THF. This clustering should also demonstrate the positioning of THF to possible binding regions. Accordingly, rmsd and diffusion analyses in reference to the co-crystallized THF structure of the MD trajectory should show patterns confirming these possible binding regions. Indeed, the rmsd analysis of the trajectory showed time intervals with low rmsd values (figure 4A) during the simulation time, which indicate the presence of THF at stable geometries during these intervals. Closer investigation of this figure shows that the average rmsd values of these time periods is high in reference to the co-crystallized THF. This indicates that THF has deviated from its original position in the x-ray structure to a new position. However, the movement of THF to a new position should be supported by diffusion of whole atoms analysis.



Mean square displacement (MSD) of THF was calculated using diffusion module of AMBER14 to find its movement from the original position in the co-crystallized structure.39 This module finds the displacement of a compound from a reference position, which is the co-crystallized position of THF, during the MD simulation based on previously specified atoms. For results to be more sensible, the diffusion averaged over all atoms of THF was calculated. To find the positional movement of THF, its displacement has been plotted in the three dimensions x, y and z axis as well as the corresponding three planes xy, xz and yz (figure 4B). The plot shows that THF has mostly moved from its original position on xz and xy planes, x and z axis. Furthermore, Figure 4B shows that THF has high MSD values at certain time intervals during the MD simulation. This displacement pattern indicates that THF has resided in certain position for long time periods of simulation time and then moved to the next position for further time interval. This shows stable binding of THF to these binding regions. To demonstrate the binding regions of THF to LSD1/CoREST complex, we superimposed the amino acids with low rmsd of x-ray structure (black), first cluster (blue) and the second cluster (green), figure 4C. It is obvious from the figure that THF has bound to different binding regions of that in x-ray structure, where it has bound to a region very close to the original position (first cluster) and distant binding region (second cluster). Figure 4D shows the relative positions of THF in first and second clusters in reference to the co-crystallized structure in LSD1/CoREST-THF complex x-ray structure.


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A

C

B

D

Figure 4. A) RMSD of THF during the MD simulation B) MSD (A2) of THF inside LSD1/CoREST binding centre during the MD simulation at x (magenta), y (green), z (light blue), xy (orange), yz (dark blue) and xz (yellow). C) Zoom-in to the binding centre of superimposed 4KUM (black), first cluster (blue) and second cluster (green) structures with the corresponding binding positions of THF. D) Positions of THF in reference to its original position (black) with three axes represented.

Binding modes of THF in binding regions of LSD1/CoREST complex active centre The 1µs MD simulation and clustering procedures of LSD1/CoREST-THF complex has resulted in two different conformers of the complex, as well as two different binding regions of THF to the complex. Accordingly, the detailed binding of THF to each binding region in each cluster and the corresponding binding modes has been investigated. Detailed THF binding modes and binding regions are important for future structure-based design to find novel selective and reversible LSD1/CoREST inhibitors. Figure 5B shows the binding of THF to the original binding region in the x-ray structure.29 In this binding region, hydrogen bonds formed between GLU389 side chain and THF-H52 (see figure 5A for THF atom numbering), backbone carbonyl oxygen of ALA639 and THF-H38, GLN188 and THF-O3, and HIS394 and THF-O3. Also, THF binds by hydrophobic interaction to PHE368, FAD, TYR591 and VAL163.29 According to the x-ray



structure, THF has a bent conformation that has intra-molecular hydrogen bonding as well as π-π interaction between benzene ring and the tetrahydro-1H-pteridin ring system.29 Investigation of this binding region showing that the hydrophilic part of THF is exposed to the surface of LSD1 chain, and the hydrophobic part is buried inside the hydrophobic binding region. In the x-ray structure, THF showed no interaction with CoREST chain.29 Figure 5C shows the binding of THF to the first cluster of the MD simulation (will be annotated as first binding region). Amino acids of the original binding region (figure 5B) are represented in ball and stick to illustrate the change in binding. In this binding region, THF forms hydrogen bond to GLU389 through THF-H53 and THF-H41. This interaction is preserved from the original binding region (figure 5B), however the binding is only through THF-H53 in the first binding region. New hydrogen bonds have formed in this binding region, which are interactions of SER393 side chain, backbone amino group of HIS394 to THF-O4, also ILE186 backbone carbonyl oxygen atom to THF-H55. In comparison to original binding region, the hydrogen bonds to PHE368 and HIS394 have been lost and three hydrogen bonds have emerged. THF forms hydrophobic interactions to ILE186, PHE368, TPP525 side chains, whereas TYR591 and VAL163 interactions in the original binding region have been lost. Furthermore, no interactions have been observed between THF and FAD molecule, which indicate that THF is distant from FAD compared to that in the original binding region. These results suggest that THF has induced a new binding region close to the original binding region. The new binding region is still within the LSD1 chain since no interactions have been observed to the CoREST chain. THF has a bent conformation similar to that in the x-ray structure. However, the intra-molecular hydrogen bond and π-π interaction have been lost, which indicate that THF has a more relaxed bent conformation with new binding forces. Inspection of the second cluster structure (figure 5D) shows new binding region which is distant from the original binding region (will be annotated as second binding region). THF forms hydrogen bonds between GLU217 side chain and THF-H52/H41 and LYS668 backbone oxygen and THF-H52. In this binding region, no hydrogen bonds have been preserved from the original binding region. This indicates that THF has bound to a new binding region.. THF forms hydrophobic interaction to PHE388, ALA639 and ALA369 in this region, which are not present in the original binding region. Also, THF has no observed interaction with FAD molecule. In


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contrast to previous two regions, THF has interactions with the CoREST chain alongside its interactions with the LSD1 chain. Moreover, THF has an extended conformation in contrast to the original and first binding regions with no intra-molecular interactions, which indicate different binding mode. To inspect the stability of these contacts (hydrogen bonds) throughout the MD simulation, nativecontact module of cpptraj of AMBER14 has been used to find distances between THF and LSD1/CoREST complex amino acids with their corresponding frequencies.39 This module finds the contacts throughout the MD trajectory frames that are found in a reference structure. Accordingly, the two represented structures were used as reference structures to find contacts through each cluster trajectory at a distance of 4Å around THF, table 1. The high frequency of these hydrogen bonds shown in table 1 demonstrates that these hydrogen bonds are stable through the MD simulation since they have high frequency for in frames of each cluster.

A

B

C

D

Figure 5. (A) Atom numbering of THF cofactor. Binding of THF to LSD1/CoREST complex in (B) x-ray structure 4KUM, (C) first cluster, and (D) second cluster. The binding region of co-crystallized THF is represented in ball and stick in first (C) and second (D) clusters to represent different binding regions of THF.



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Table1. Contacts (hydrogen bonds) between THF and amino acid residues of LAD1/CoREST complex for frames of each cluster trajectory as calculated by nativecontact in cpptraj of AMBER14, with their corresponding frequencies.

Cluster2

Cluster1 Average Contacts

Frequency

distance

Average Contacts

Frequency

(Å)a

a)

distance (Å)a

GLU389 to THF-H53

0.94

3.35

LYS668 to THF-H52

0.76

2.89

GLU389 to THF-H41

0.83

3.18

GLU217 to THF-H52

0.85

3.13

SER393 to THF-O4

0.28

3.54

GLU217 to THF-H41

0.72

3.22

HIS394 to THF-O4

0.93

3.03

ILE186 to THF-H55

0.87

2.98

Distance between heavy atoms of the hydrogen bond acceptor and donor.

Binding free energy analysis The binding free energy of THF to LSD1/CoREST complex has been calculated in both binding modes. It was calculated using molecular mechanics combined with Poisson–Boltzmann and surface area continuum solvation (MMPBSA). 51 First 50 frames from each cluster were used for MMPBSA calculation with ionic strength was set to 0.1 M, a solute dielectric constant of ε = 1.0 and a solvent dielectric constant of ε = 80.0. The binding free energy and its corresponding components obtained from the MMPBSA for THF in both binding modes are reported in Table 2. The results show that the two binding modes in their corresponding binding regions are favourable from the negative ∆Gbindig values. The favourable binding of THF to the complex is driven by the van der Waals contributions (Evwd) and the salvation nonpolar contributions (Gsolvnon-polar).

These two components favour the binding at first binding region, Evwd = -36.80 kcal/mol

and Gsolv-non-polar= -5.25, and the second binding region Evwd = -29.80 kcal/mol and Gsolv-non-polar= -5.14.


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Table2. Free energy of binding and its energy components (Kcal/mol) for THF in both binding modes. THF binding region

Eelec

Evwd

Gsolv-polar

Gsolv-non-polar

∆Gbindig

First binding region

-33.77

-36.80

57.43

-5.25

-18.39

Second binding region

-93.99

-29.80

100.20

-5.14

-28.74

The electrostatic component of the binding free energy is an important contributor in the ligandreceptor complex formation. The involvement of electrostatic interaction with water disfavours the formation of the complex, since this unfavourable change in the electrostatics of solvation needs to be compensated by the favourable electrostatics within the ligand–receptor complex.52 The values for the total electrostatic component (Eelec and Gsolv-polar) in table 2 for both binding regions suggest unfavourable binding since the total electrostatic component is positive, +23.66 kcal/mol for the first binding region and +6.21 kcal/mol for the second binding region. Table 2 shows that Gsolv-polar is more positive for the second binding region, which is expected since this is closer to the surface. Also, the Eelec is more negative for the second binding region since it is mostly composed of hydrophilic amino acids. Discussion The molecular dynamics simulation in this study is based on 4KUM x-ray structure that contains THF cofactor bound to LSD1/CoREST-FAD complex. Purposely, 4KUM structure was investigated in this study in order to utilise its co-crystallized THF cofactor to find possible binding regions. Furthermore, the conformational change of the complex upon binding of THF was explored. LSD1/CoREST complex has been investigated by MD in different research studies.25,28,53,52 All these studies focused on the MD of the unbound LSD1/CoREST complex and that bound to the histone H3 tail to find possible conformational existence of this complex. According to previous studies, LSD1/CoREST complex has two dominant conformational transitions upon histone H3 tail substrate binding, the open and closed conformations.25,54 This conformational transition, which is controlled by H3 tail binding, is important for chromatin and protein binding.25,54 In our study, THF, which should properly bind to the active centre of the complex, was utilised as the natural cofactor for this enzyme complex. Although the substrate of the enzyme complex (histone H3 tail) has been used in previous MD simulations,25,28 our



findings also support the open-closed conformation transition upon binding of THF. According to our 1µs MD simulation, LSD1/CoREST complex has two dominant conformational clusters upon THF binding, figures 1, 2 and 3. Movie S1, which is created from first PC, explains the close conformation upon binding of THF to the complex. Movie S2, which is created from the second PC, describes the opening of the complex upon unbinding of THF from its original position to the surface position. THF binding showed that LSD1 domain is flexible at its tower region while CoREST domain is rigid. According to Baron, R et al. study, H3 tail binding induces rigid body movement of AOD (figure 3) with respect to the tower.54 Our findings showed that this movement is controlled by the flexibility of the tower region of LSD1 chain (see movies S1 and S2). According to these results, the binding of THF induces the close conformation of LSD1/CoREST (movie S1), which might be a requirement for the assembly of histone H3 tail substrate and THF cofactor in the active centre of the complex for the enzymatic mechanism. The two conformational structures induced by the binding of THF are correlated with its position in the active centre of the complex. According to the 1µs MD simulation, THF binds to two positions in the active centre, figure 4 and table 1. The first position (first cluster) corresponds to the binding of THF to a region close to the original binding region. This is inferred from the stable rmsd and diffusion values at this position, figure 4. This binding position corresponds to the closed conformation of the complex, where THF is close to FAD at the core of LSD1/CoREST complex to aid the acceptance of CH2 group from tail H3 methylated lysine residue, see movie S4.29 The second position (second cluster, figure 4) is located at the surface of the complex and has a stable binding to THF at its entrance to the active centre (movie S3). This is evident from the rmsd and diffusion patterns of THF. This position corresponds to the open position of LSD1/CoREST complex, see figure 3, table1 and movie S2. Although the conformational change of LSD1/CoREST complex has been investigated in different studies, the binding regions emerged upon binding of the substrate or the cofactor have not been addressed. Also, the binding modes and binding amino acids have not been investigated previously. However, Robertson, J.C. et al. has investigated the expansion of the druggable space of LSD1/CoREST complex based on computational methods like FTMap and SiteMap.28,55 These algorithms are able to locate druggable site within the protein according to a user defined


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parameters with good confidence.55 Furthermore, these algorithms succeeded to find different binding sites even of the ephemeral states of the receptor.28 However, amino acid residues that are predicted to be involved in the binding site of the complex are not based on an interaction between a ligand and amino acid residues. Accordingly, the use of a cofactor that is known to bind appropriately to the protein binding centre should confer more information about the nature of binding regions. Our results showed that THF cofactor bind to two binding regions associated with the closed and open conformations of the complex as discussed above. In the first binding region THF binds in its bent conformation similar to that of the co-crystallized structure. This binding region is close to FAD molecule and has both hydrophobic and hydrophilic binding properties. The second binding region is mainly hydrophilic and involves amino acid residues from the CoREST chain. This binding region is potentially evolved at the entry of THF to the active centre of the complex, where THF binds in its extended form (movie 4). Amino acids involved in the interaction in both THF positions are represented in the results section. These binding regions with the corresponding complex conformation and amino acids bound to THF will be used in future for docking studies to find inhibitors of this enzyme complex. Based on the results represented here, we propose that THF has a stable binding to a binding region at its entrance to the active centre of LSD1/CoREST complex and involved both LSD1 and CoREST chains, tables 1 and 2. Then, it enters the active centre of the complex inducing the closed conformation of the complex to be close to FAD for its function as carbon acceptor from the substrate. Conclusions LSD1/CoREST complex is an enzyme that is involved in the demethylation of lysine of histone H3 tail. This enzyme could be targeted to suppress tumour cell growth. We presented 1µs MD simulation for this complex which contains THF cofactor bound to LSD1/CoREST-FAD to find conformational changes of the complex upon THF binding. Also, the THF binding regions and binding modes have been investigated for future structure based drug design. According to the MD simulation results, THF enters the active centre of the complex and forms stable binding region, where the complex is in its open conformation. This binding region is mainly hydrophilic. Then, THF binds stably to the other binding region in the core of the



LSD1/CoREST complex, which corresponds to the closed conformation of the complex. This binding site has hydrophilic and hydrophobic properties.

ASSOCIATED CONTENT Supporting Information Additional figures describe the stability of the MD simulation, Figure S1. Movies S1 to S4 could be found as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgment The authors would like to thank Abd Al Hamid Shoman Foundation for funding this research according to the agreement number 13/2015.


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For Table of Contents Only

Exploring the Active Centre of LSD1/CoREST Complex by Molecular Dynamics Simulation utilizing its Co-Crystallized Cofactor Tetrahydrofolate as a Probe

Waleed A. Zalloum and Hiba M. Zalloum


CoREST Complex by

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