Interdiscip Sci Comput Life Sci (2015) 7: 1–10 DOI: 10.1007/s12539-014-0228-7
QM/MM MD and Free Energy Simulation Study of Methyl Transfer Processes Catalyzed by PKMTs and PRMTs 1
2
Yuzhuo Chu1∗ , Hong Guo2
(School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China) (Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996 and UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6164)
Received 16 July 2014/ Revised 19 September 2014/ Accepted 17 October 2014
Abstract: Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here we review the results of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes. Key words: methyl transfer, PKMT, PRMT, product specificity, QM/MM MD, free energy simulations, reaction mechanism.
1 Overview Methyl transfer processes play important roles in many different biological functions. One type of methyl transfer processes, histone lysine methylations occurring at the histone tails govern many important biological events, including heterochromatin formation, Xchromosome inactivation, transcriptional silencing and activation (Jenuwein 2006; Martin and Zhang 2005). These methylations are catalyzed mostly by the SET domain protein lysine methyltransferases (PKMTs) through transferring methyl groups from S-adenosylL-methionine (AdoMet) to the amino group of a lysine residue on the histone, leading to a methylated lysine residue and the cofactor byproduct S-adenosylL-homocysteine (AdoHcy) (Trievel 2004). A range of lysine (K) residues in different histones, including K4, K9, K27, K36 and K79 in histone H3, K20 in histone H4, etc have been found to be the methylation targets. The SET domain PKMTs may be divided into the following families, SUV39, SET1, SET2, EZ, RIZ, SMYD, SUV4-20, and also orphan members like SET7/9 and SET8. These members contain a similar SET domain and similar motifs surrounding the SET domain (Dillon et al. 2005). One important property of PKMTs is their product specificity, which refers to their dif∗
Corresponding author. E-mail: [email protected]
ferent ability to add one, two or three methyl groups to the target lysine (Cheng et al. 2005; Xiao et al. 2003). Specific methylation states are enriched in different regions of genes, recognized by different methyllysine binding proteins and lead to different subsequent biological events (Li et al. 2007; Taverna et al. 2007). Thus it is important to understand the mechanism underlying the product specificity of PKMTs. A specific Tyr/Phe switch position within the lysine binding site has been indicated to control the product specificity of PKMTs. This position is frequently occupied by a tyrosine in the mono-methyltransferases and by a phenylalanine or another hydrophobic amino acid in the diand tri-methyltransferases (Collins et al. 2005; Zhang et al. 2003). The mutations of other important activesite residues beyond the Tyr/Phe switch could also alter the product specificity of PKMTs (Del Rizzo et al. 2010; Wu et al. 2010; Zhang et al. 2003). However, the mechanism of how the Tyr/Phe switch and other activesite mutants alter the enzyme product specificity is still unclear due to the limitations of experimental studies. Another important methyl transfer process, protein arginine methylation, occurs in abundant eukaryotic proteins and modulates different cell signaling pathways, including gene transcription, RNA splicing, signal transduction, cell growth and proliferation (Bedford and Clarke 2009). These methyl transfer processes are catalyzed by the family of protein arginine methyltransferases (PRMTs) through the transfer of methyl
2
groups from AdoMet to the guanidine group of arginine residues in protein substrate (Lee et al. 1977). There are two major types of PRMTs which catalyze the formation of different products. Both types of enzymes catalyze the formation of ω-N G -monomethylarginine (MMA). Type I PRMTs catalyze the formation of asymmetric ω-N G , N G -dimethylarginine (ADMA), while type II PRMTs catalyze the formation of symmetric ω-NG , N G -dimethylarginine (SDMA) (Di Lorenzo and Bedford 2011). Different dimethylated product (ADMA or SDMA) might lead to different biological consequences (Wysocka et al. 2006), thus it is very important to understand the basic mechanism underlying the methylation processes and the product specificity of PRMTs. Molecular dynamics (MD) simulations based on molecular mechanics (MM) force field could be performed to obtain the structural information of the reactant complex. However, the description of the electron re-distribution during covalent bond breaking and making process requires a quantum mechanics (QM) potential (Lin and Truhlar 2007). Although high-level ab initio calculations (e.g., B3LYP/6-31G** or MP2/631G**) have been widely used for simple systems, such as proton sponges (Guo and Salahub 2001), polyacetylene (Guo and Paldus 1997) and peptides with carbon hydrogen bonds (Guo et al. 2004), these approaches are still too time consuming for studying enzyme-catalyzed reactions. The hybrid QM/MM MD simulations combine the accuracy of QM with the low computational cost of MM by treating only the reacting part of the systems quantum mechanically and the other part molecular mechanically (Warshel and Levitt 1976). These approaches have been utilized to study enzyme reactions in different biological systems (Guo et al. 2006; Guo et al. 2005; Hu et al. 2008; Hu et al. 2008; Wang et al. 2007; Xu et al. 2007; Xu et al. 2007; Xu et al. 2006; Xu et al. 2010; Yao et al. 2011; Zhang and Bruice 2008). The energetic results of SCC-DFTB and high-level B3LYP/6-31G** methods for the methyl transfer in a simple model system that resembles the methyl transfer in PKMTs and PRMTs were compared to understand the performance of the semi-empirical method in these reactions (Chu et al. 2013; Guo and Guo 2007; Xu et al. 2009). For PKMTs, although the SCC-DFTB optimized geometries along the reaction pathway seemed to be rather close to those from B3LYP/6-31G**, there are some systematic deviations of the SCC-DFTB method in the description of the energetics of the methyl transfer. To correct the errors due to the deficiency of SCC-DFTB method, the empirical correction (Guo and Guo 2007; Xu et al. 2009) was applied to the free energy curves obtained from the potential of mean force simulations. The energy curves from the corrected SCC-DFTB and B3LYP/6-31G** were very close, supporting the use of the approach
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
with the empirical correction. For PRMTs, the potential energy function obtained from SCC-DFTB is quite similar to that obtained from the B3LYP/6-31G** calculations. Therefore, no empirical correction seems to be necessary for this reaction (Chu et al. 2013). In this review, we will discuss the applications of QM/MM MD and free energy simulations in the study of methyl transfer processes catalyzed by PKMTs and PRMTs (Chu et al. 2013; Chu et al. 2010; Chu et al. 2012; Guo and Guo 2007; Xu et al. 2009). The reaction mechanism and the mechanism underlying the special properties of these enzymes, such as the product specificity, are investigated in these computational studies.
2 QM/MM MD and free energy simulations of the methyl transfers catalyzed by PKMTs 2.1
SET7/9
Initially identified as a histone3 lysine 4 (H3-K4) specific methyltransferase, SET7/9 could also regulate the functions of non-histone substrates through site-specific methylation (Chuikov et al. 2004). The Tyr/Phe switch mutant Y305F alters the enzyme from a monomethyltransferase to a di-methyltransferase. Another active-site mutant, Y245A alters the enzyme to a trimethyltransferase with weaker activity (Del Rizzo et al. 2010). In the crystal structure of SET7/9 in complex with substrate histone peptide and cofactor Sadenosylhomocysteine (AdoHcy) (PDB ID: 1O9S), the substrate nitrogen is aligned with the active methyl group of AdoMet via hydrogen bonding to the hydroxyl group of Y245 and the oxygen atom of an active-site water molecule. This water molecule is further stabilized in the active site via hydrogen bonding to the hydroxyl group of Y305 (Xiao et al. 2003). The free energy barriers of the methyl transfer processes were calculated from QM/MM MD and free energy simulations along the reaction coordinate [R = r(CM −Sδ ) – r(CM −Nε )] (Fig. 1) (Guo and Guo 2007). The calculated free energy barrier of the second methyl transfer process in wild type (wt) SET7/9 is about 5 kcal/mol higher than that of the first methyl transfer process. The higher free energy barrier of the second
r2
H3K4
Fig. 1
Nξ
r2 rI
CM Sδ
A doMet AdoMet
θ θ
rI r2
The reaction coordinate for calculating the free energy barriers of the methyl transfer process in SET7/9 (PKMTs) is R = r(CM −Sδ ) – r(CM −Nξ ). Figure adapted from (Guo and Guo 2007).
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
3
bonding to Y245 and also an active-site water molecule. However, the substrate could not be well aligned with AdoMet in the second methyl transfer of wt SET7/9. In this case, the substrate is pushed away from AdoMet in the more crowded active site after it accepts one methyl group. the substrate is pushed away from AdoMet since the active site becomes more crowded after it accepts one methyl group. As for the second methyl transfer in Y305F, there is more space around the substrate since the active-site water molecule could dissociate, leaving the substrate well aligned with AdoMet and making this methyl transfer process possible to proceed (Guo and Guo 2007).
methyl transfer process makes this process difficult to proceed than the first methyl transfer. In contrast to wt SET7/9, the calculated free energy barriers of the first and second methyl transfer processes in Y305F are similar, consistent with the fact that Y305F alters the enzyme to a di-methyltransferase (Guo and Guo 2007). The reactant complex conformations for the methyl transfer processes were also obtained from QM/MM MD simulations (Fig. 2). The active-site structures of the reactant complex of the simulated methyl transfer processes indicate that for the first methyl transfer in wt SET7/9, the target nitrogen is well aligned with the active methyl group of AdoMet via hydrogen
N265 N265
Y245
o1
H3K4me
o1 2.3 2.9 h1 h2 3.6 2.6
1.8
h 4.0
Y245
Y305
1.8
Y335 W1 A295
AdoMet
Y245 AdoMet
A295
Y305 (b)
G264 h 2.2 3.2
H293
1.8 H293
Y335 W1 A295
F305
AdoMet
(c)
The active-site structures of the reactant complex for the methyl transfers in wt SET7/9 and Y305F mutant obtained from QM/MM MD simulations. (a) The first methyl transfer in wt SET7/9. (b) The second methyl transfer in wt SET7/9. (c) The second methyl transfer in Y305F. Figures adapted from (Guo and Guo 2007).
The calculated free energy barriers of the first, second and third methyl transfer processes in Y245A mutant are similar, making all these processes possible to proceed. Also, the barrier of the first methyl transfer process is about 2 kcal/mol higher than that of the first methyl transfer in wt SET7/9, consistent with the fact that the methylation catalyzed by Y245A mutant is less efficient. The reactant complex conformations of all three methyl transfer processes in Y245A indicate that the substrate nitrogen is well aligned with AdoMet. The mutation of Y245 to alanine generates more free space in the active site, making it possible for the substrate to accept three methyl groups (Yao et al. 2012). 2.2
H3K4me
o2
(a)
Fig. 2
N265
H293
o2 Y335 W1
N265
G264
N264 H3K4
N265
N265
DIM-5
DIM-5 catalyzes the tri-methylation of H3-K9 and marks chromatin regions for DNA methylation (Tamaru et al. 2003). The crystal structure of DIM5 in complex with AdoHcy and H3 peptide (PDB ID: 1PEG) indicates that the substrate lysine is stabilized in the active site via van der Waals contact between its methylene group and the aromatic side chains of F206, F281, Y283 and W318 in DIM-5 (Zhang et al. 2003). The substrate amino group also forms hydrogen bond with the hydroxyl group of another active-site residue
Y178. The mutation of the Tyr/Phe switch, F281Y, does not reduce the overall catalytic activity of DIM-5 but alters it to a mono-/di-methyltransferase (Zhang et al. 2003). QM/MM MD and free energy simulations were applied in DIM-5 to study the origin of its product specificity and its changes due to mutations (Xu et al. 2009). The calculated free energy barriers of the first, second and third methyl transfer processes in DIM-5 are similar, consistent with the fact that DIM-5 is a tri-methyltransferase. For F281Y mutant, the calculated free energy barrier of the second methyl transfer process is about 3 kcal/mol higher than the first transfer and the barrier of the third methyl transfer process is about 8 kcal/mol higher than the first transfer. The higher free energy barrier of the second and third methyl transfer processes in F281Y mutant could stop the further methylation process. These results suggest that the relative free energy barriers for the methyl transfers might be the energetic factors controlling the enzyme product specificity. The reactant complex conformations for the methyl transfer processes in DIM-5 and its F281Y mutant were also obtained from QM/MM MD simulations (Xu et al. 2009). The comparison of the reactant complex conformation of the third methyl transfer process in DIM-5 and F281Y indicates that the electron lone pair
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Interdiscip Sci Comput Life Sci (2015) 7: 1–10 Y204
V203
Y204
V203 Y178 Y178 3.0
H3K9(me)2 4.6
1.8
1.8 Y283
H3K9(me)2 R238 W1
R238
Y283 I240
F281
Y281 W1
AdoMet AdoMet
(a)
Fig. 3
(b)
The active-site structures of the reactant complex for the third methyl transfer processes in wt DIM-5 and F281Y mutant obtained from simulations. (a) The third methyl transfer in wt DIM-5. (b) The third methyl transfer in F281Y mutant. Figures adapted from (Xu et al. 2009).
on the Nε atom of the target di-methylated lysine is well aligned with the active methyl group of AdoMet in wt DIM-5 (Fig. 3), but not in the F281Y mutant. These results suggest that the formation of reactive configuration in the reactant complex might be used as a predictor for the product specificity. Similar as the case of SET7/9, a significant difference between the active-site structure of the reactant complex in the third methyl transfer of DIM-5 and F281Y is the location of an active-site water molecule. In DIM-5, this water molecule is able to dissociate from the active site, leaving more space around the target nitrogen to form well alignment with AdoMet. As for F281Y, this water molecule is stabilized in the active site via hydrogen bonding with the hydroxyl group of Y281. The active-site space around the target nitrogen becomes more crowded and thus the target nitrogen is pushed further away from the active methyl group of AdoMet. These structural information obtained from simulations suggest that the dissociation of this active-site water molecule plays an essential role in the determination of the product specificity of DIM-5 and its Y281F mutant (Xu et al. 2009). The mutation of Y281 to tryptophan abolishes the methylation activity of DIM-5 completely (Zhang et al. 2003). Consistent with this fact, the calculated free energy barrier of the first methyl transfer of Y281W is about 6 kcal/mol higher than that of wt DIM-5. The reactant complex conformation of Y281W first methyl transfer also indicates that the target nitrogen is not well aligned with the active methyl group of AdoMet, possibly due to the steric hinder caused by the sidechain of W281, which occupies more space (Xu et al. 2009). 2.3
AdoMet I240
strate H3K20 and cofactor AdoHcy (PDB ID: 1ZKK) (Couture et al. 2005), the substrate lysine is oriented towards the cofactor via hydrogen bond interactions with the hydroxyl group of an active-site residue Tyr245 and also an active-site water molecule (W1). This water molecule is further stabilized in the active site via hydrogen bonding to Tyr334 hydroxyl group and the carbonyl oxygen of Gly-294 and Ile-297 (Fig. 4). The mutation of the Tyr/Phe switch of SET8 (Y334F) could alter this enzyme to a di-methyltransferase (Couture et al. 2005). The structural observations of Y334F mutant in complex with histone H4 bearing unmodified, mono-methylated, and di-methylated status suggest that Y334F mutant might alter the product specificity of SET8 through affecting the dissociation ability of an active-site water molecule (Couture et al. 2008). QM/MM MD and free energy simulations were applied to reveal the methyl transfer processes catalyzed by SET8 and its Y334F mutant and to study the origin of their product specificity (Chu et al. 2010). The cal-
Tyr271 K20
ADY Tyr245
Gly2944
SET8
SET8 is implicated in cell-cycle-dependent transcriptional silencing (Nishioka et al. 2002) and mitotic regulation in metazoans (Karachentsev et al. 2005). It catalyzes the specific mono-methylation of H4-K20. In the active-site structure of SET8 in complex with sub-
Tyr336 3 W1
Ile297 Tyr334
Fig. 4
The active-site structure of SET8 in complex with AdoHcy and H3K20 (PDB ID: 1ZKK) (Couture et al. 2005).
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
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the active-site structures of the reactant complex for the first methyl transfer of SET8, the first and second methyl transfers of Y334F mutant, the lone pair of electrons on the target Nε atom is well aligned with the active methyl group of the cofactor AdoMet. However, in the active-site structures of the reactant complex for the second methyl transfer of wt SET8 and the third methyl transfer of Y334F mutant, the lone pair of electrons on the target Nε atom is not well aligned with the active methyl group of AdoMet. These results suggest that the formation of reactive configuration in the reactant complex obtained from MD simulations could be used as a predictor of the product specificity. The reactions will be less efficient if the reactive configuration could not be formed in the reactant complex, since additional energy cost will be required to generate the reactive configuration first. In the active-site structure of the reactant complex for the second methyl transfer in SET8, the active-site water molecule is stabilized via hydrogen bonds interactions with the hydroxyl group of Tyr334. The active site of the reactant complex for this methyl transfer process is too crowded, thus the target nitrogen and the active methyl group of AdoMet could not form well alignment. In the active-site structure of
culated free energy barrier of the second methyl transfer process of wt SET8 is about 6.5 kcal/mol higher than that of its first methyl transfer process. As for the Y334F mutant, the free energy barriers of the first and second methyl transfer processes are similar with that of the first methyl transfer in wt SET8. However the free energy barrier of its third methyl transfer process is about 9 kcal/mol higher than the first methyl transfer process in wt SET8. The calculated free energy barriers of the methyl transfer processes in wt SET8 and its Y334F mutant are consistent with their product specificity, which suggest that the high free energy barrier of certain methyl transfer process (for instance, the second methyl transfer in wt SET8 and the third methyl transfer in Y334F mutant) could stop the further methylation. Y334F mutant could alter the enzyme to a di-methyltransferase since it lowers the free energy barrier of the second methyl transfer process (Chu et al. 2010). The active-site structures of the reactant complex for the methyl transfers in wt SET8 and Y334F mutant obtained from the simulations indicate that their product specificity are already reflected from their reactant complex conformations (Chu et al. 2010) (Fig. 5). In Tyr271
Tyr271 Tyr245
Tyr245
3.0
2.4 3.0 1.8 H4K20 3.2
1.8
4.5 H4K20
5.0 5.0
W1
W1 AdoMet
Tyr334
AdoMet
Tyr334 (a)
(b)
Tyr271
Tyr271
Tyr271
Tyr245 Tyr245 2.2 3.0
2.9 1.8 3.0 H4K20 2.7 5.0 W1 Phe334
1.8
4.6
H4K20
1.8
11.0 H4K20 W1 AdoMet AdoMet
Phe334 (c)
Fig. 5
Tyr245
(d)
Phe334
AdoMet (e)
The active-site structures of the reactant complex for the methyl transfers in wt SET8 and Y334F mutant obtained from simulations. (a) The first methyl transfer in wt SET8. (b) The second methyl transfer in wt SET8. (c) The first methyl transfer in Y334F. (d) The second methyl transfer in Y334F. (e) The third methyl transfer in Y334F. Figures adapted from (Chu et al. 2010).
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the reactant complex for the second methyl transfer in Y334F mutant, this water molecule is able to dissociate from the active site since its hydrogen bond interactions with the hydroxyl group of Tyr334 is lost due to its mutation to Phe. The dissociation of this active-site water molecule generates more space around the target nitrogen and allows it to form well alignment with the active methyl group of AdoMet. 2.4
GLP
G9a-Like-Protein (GLP) and its closely related homolog G9a are the major mammalian H3-K9 methyltransferases that target euchromatic regions and essential for murine embryogenesis (Tachibana et al. 2002). They form a heteromic complex and work cooperatively in euchromatin to exert H3-K9 methyltransferase function in vivo (Tachibana et al. 2005). The Tyr/Phe switch sites of GLP and G9a are occupied by a Phe and the mutation of this site in G9a (F1152Y) alters the enzyme from a di-methyltransferase to a mono-methyltransferase. The equivalent residue in GLP is F1209. In addition to the single-residue mutant at the Tyr/Phe switch site which could alter the product specificity of G9a, the mutation of another active-site residue, Y1206F, could alter it to a
Table 1
tri-methyltransferase. The equivalent residue in GLP is Y1124 (Collins et al. 2005) (Table 1). The calculated free energy barriers of the methyl transfer processes in wt GLP, its F1209Y, and Y1124F mutants are consistent with their product specificity (Chu et al. 2012). For wt GLP, the calculated free energy barrier of the first and second methyl transfer processes are similar. Whereas the barrier of the third methyl transfer process is about 9 kcal/mol higher than that of the first methyl transfer. For the F1209Y mutant, the free energy barrier of the second methyl transfer process is increased to about 4 kcal/mol higher than that of the first methyl transfer process. The high energy barrier of this step might stop the further methylation and thus F1209Y is a mono-methyltransferase. As for the Y1124F mutant, the free energy barrier of the third methyl transfer process is also lowered to be similar with the first methyl transfer, which allows the third methylation process to proceed. These results are consistent with the conclusions from previous studies of other PKMTs that the free energy barrier of the methyl transfer process might determine the energetic origins of the product specificity of these enzymes and their mutants (Chu et al. 2012).
The product specificity of wt G9a and its active-site mutants (with the equivalent mutants of GLP listed)
wt G9a
F1152Y of G9a (F1209Y of GLP)
Y1206F of G9a (Y1124F of GLP)
Di-methyltransferase
Mono-methyltransferase
Tri-methyltransferase
As suggested from previous PKMTs studies, the product specificity of GLP and its mutants could also be reflected from the reactant complex conformations obtained from the simulations (Chu et al. 2012). In the first, second methylations of wt GLP, the first methylation of F1209Y and the first, second and third methylations of Y1124F, the lone pair of electrons on the target Nε atom is well aligned with the active methyl group of the cofactor AdoMet in the reactant complex, consistent with the fact that these methylation processes are efficient. As for the methylations which are not efficient, such as the third methylation of wt GLP, the second, third methylations of F1209Y, the target nitrogen could not form well alignment with AdoMet. The structural information of the transition-state complex for the second methylation in wt GLP obtained from the free energy simulations indicates that the active-site water molecule could dissociate from the active-site during this methylation process. This generates more space around the target nitrogen to allow it to form well alignment with AdoMet. As for the F1209Y mutant, the structural information of the transition-state complex
for the second methylation indicates that this activesite water molecule is stabilized via hydrogen bonding to the hydroxyl group of Y1209. The active-site space around the target nitrogen is too crowded thus it could not be well aligned with AdoMet. The third methylation in GLP could not proceed since the active-site space around the target nitrogen is too crowded after two methyl groups are added. Whereas the loss of the hydroxyl group due to Y1124F mutation generates more active-site space, facilitates the well alignment of the target nitrogen and AdoMet in its third methylation step (Fig. 6). The computational results of GLP, its F1209Y, and Y1124F mutants suggest that the activesite space around the target nitrogen is an important factor for the formation of reactive configurations and the methylation activity (Chu et al. 2012).
3 QM/MM MD and free energy simulations of the methyl transfer processes catalyzed by PRMT3 QM/MM MD and free energy simulations were
Interdiscip Sci Comput Life Sci (2015) 7: 1–10 Tyr1124
Tyr1142 Tyr1142 Tyr114 1442 1.88
Phe1124
4.5
H3K9me2 3K9me2
H3K9 AdoMet
Phe1209 he1209
Phe12099
(a)
Fig. 6
3.0 1.88
AdoMet (b)
The active-site structures of the reactant complex for the third methyl transfer processes in wt GLP and Y1124F mutant obtained from simulations. (a) The third methyl transfer in wt GLP. (b) The third methyl transfer in Y1124F. Figures adapted from (Chu et al. 2012).
Peptidyl-Arg θ Nη1 Nη2
Fig. 7
Sδ AdoMet
CM r(CM−Nη2)
The reaction coordinate for calculating the free energy barriers of the methyl transfer process in PRMT3 is R = r(CM − Sδ ) – r(CM − Nη ). Figure adapted from (Chu et al. 2013).
7
active-site arginine residue. Thus the positive charge of the guanidine group is redistributed towards the amino group near Glu335 and leaving the Nη2 atom to be the possible nucleophile (Chu et al. 2013; Zhang and Cheng 2003; Zhang et al. 2000). The possible general base during the catalysis has also been discussed in PRMTs (Rust et al. 2011; Zhang et al. 2000), whereas the knowledge is very limited. A His-Asp relay system has been suggested to function as the general base (Zhang et al. 2000). However, the location of the His residue (>6˚ A from the substrate guanidine group) makes it less possible to function as the general base; the mutagenesis study also did not support its role as the general base (Rust et al. 2011). QM/MM free energy simulation results suggest that the conserved Glu326 might function as the general base. The transition-state structures obtained from the simulations indicate that the proton on the attacked nitrogen has transferred to the carboxyl group of Glu326 (Fig. 9(a)) (Chu et al. 2013). 2D free energy simulation was also performed to reveal the second methylation process and to further explore the relationship between the methyl transfer and proton transfer processes (Fig. 9(b)). The simulation results suggest that the proton on the Nη2 atom is transferred to the carboxyl group of Glu326 near the transition state and the proton and methyl transfer processes are somehow concerted (Chu et al. 2013).
4 Summary performed on one Type I PRMT, PRMT3, to study its reaction mechanism and also the origin of its product specificity which leads to the formation of ADMA product instead of the SDMA product. The calculated free energy barriers (along the reaction coordinate [Rx = r(CM − Sδ ) – r(CM − Nη )]) indicate that for both the first and second methyl transfer processes, the barriers of the methylation on the Nη1 atom of the target arginine are much higher than that of the Nη2 atom (about 7-8 kcal/mol higher). This suggests that both methyl groups would be added to the Nη2 atom and the ADMA product would be generated (Chu et al. 2013). The active-site structures of the reactant complex for the first and second methylation processes obtained from simulations also indicate that the Nη2 atom is well aligned with the active methyl group of AdoMet (Fig. 8) (Chu et al. 2013). The substrate arginine is stabilized in the active site via several active-site interactions, for instance, hydrogen bonding to the conserved “double E” residues (Glu326 and Glu335). The chargecharge interactions between Glu326 and AdoMet also helps to direct the active methyl group towards the Nη2 atom. The negative charge of Glu326 is greatly reduced through interactions with AdoMet and a nearby
QM/MM MD and free energy simulations were performed to study the catalytic mechanism and product specificity of several PKMTs and PRMTs. The computational results suggest that the dissociation ability of an active-site water molecule determines the product specificity of the Tyr/Phe switch of PKMTs. The active-site space around the target nitrogen also controls the product specificity of other PKMT mutants. The calculated free energy barriers of the methyl transfer processes are consistent with their efficiency, suggesting that these barriers might be the energetic control of the product specificity. The formation of reactive configuration in the reactant complex obtained from the simulations are also consistent with the reaction efficiency, suggesting that they could be used as a predictor for the methylation activity. As for the PRMTs, the calculated free energy barriers of the methyl transfer processes suggest that both methyl groups would be added to the Nη2 atom, thus generating the ADMA product. The reactant complex conformations also suggest that for both methyl transfer processes, the Nη2 atom is well aligned with the active methyl group of AdoMet. The computational results also suggest that one of the “double E” residues, Glu326, might function as the general base of the re-
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Interdiscip Sci Comput Life Sci (2015) 7: 1–10
Sub
Sub R236
2.5±0.2
OE1 3.1±0.3 HH21 Nη2 E326 3.2±0.2
Nη1
3.6±0.1
HH22 2.1±0.3
1.7±0.1
2.0±0.2
2.5±0.4
R236
E335
HH22 1.7±0.1
1.9±0.3
OE1
3.2±0.2
1.7±0.1
Nη1 1.9±0.4 11.7°±6.5
Y221
37.4°±6.6
Y221
1.7±0.1 2.1±0.3
(a)
E326 ADM (b)
1.9±0.3
HH21 Nη2 R236
1.9±0.1 HH22 1.8±0.2
E335
1.8±0.2
Nη1 Y221 2.2±0.04 1.8±0.2
OE1 1.8±0.2 E326 2.0±0.3
2.0±0.04
ADM (a)
Ry=r(HH22...Nη2)−r(HH22...OE1) (Å)
The active-site structures of the reactant complex for the methyl transfers in PRMT3 obtained from simulations. (a) The first methyl transfer. (b) The second methyl transfer. Figures adapted from (Chu et al. 2013).
Sub
Fig. 9
E335
Nη2
ADM
Fig. 8
1.9±0.3
1.4
0.7
0
−1
0 1 Rx=r(CM...Sδ)−r(CM...Nη2) (Å) (b)
(a) The active-site structure of the transition state complex for the first methyl transfer in PRMT3 obtained from simulations. (b) 2D free energy contours for the methyl transfer from AdoMet to methyl arginine and the proton transfer between methyl arginine and Glu326. Figures adapted from (Chu et al. 2013).
action. The methyl transfer and proton transfer processes are somehow concerted as suggested from the 2D free energy simulations. In future studies, QM/MM MD and free energy simulation approaches could be applied to study the properties of other enzyme-catalyzed methyl transfer processes and also other important biological reactions.
References [1] Bedford MT, Clarke SG (2009) Protein arginine methylation in mammals: who, what, and why. Mol Cell 33 (1): 1-13. doi: 10.1016/j.molcel.2008.12.013 [2] Cheng X, Collins RE, Zhang X (2005) Structural and sequence motifs of protein (histone) methylation enzymes. Annu Rev Biophys Biomol Struct 34: 267-94. doi: 10.1146/annurev.biophys.34.040204.144452 [3] Chu Y, Li G, Guo H (2013) QM/MM MD and free energy simulations of the methylation reactions catalyzed by protein arginine methyltransferase PRMT3. Can J Chem 91 (7): 605-612. doi: 10.1139/cjc-2012-0483 [4] Chu Y, Xu Q, Guo H (2010) Understanding Energetic Origins of Product Specificity of SET8 from QM/MM Free Energy Simulations: What Causes the
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9 the methylation state specificity? An ab initio QM/MM molecular dynamics study. Abstr Pap Am Chem S 235 [21] Hu P, Wang S, Zhang Y (2008) How do SET-domain protein lysine methyltransferases achieve the methylation state specificity? Revisited by ab initio QM/MM molecular dynamics simulations. J Am Chem Soc 130 (12): 3806-3813. doi: 10.1021/ja075896n [22] Jenuwein T (2006) The epigenetic magic of histone lysine methylation. Febs j 273 (14): 3121-35. doi: 10.1111/j.1742-4658.2006.05343.x [23] Karachentsev D, Sarma K, Reinberg D, Steward R (2005) PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis. Gene Dev 19 (4): 431-435. doi: 10.1101/gad.1263005 [24] Lee HW, Kim S, Paik WK (1977) Sadenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme. Biochemistry 16 (1): 78-85 [25] Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. Cell 128 (4): 707-19. doi: 10.1016/j.cell.2007.01.015 [26] Lin H, Truhlar DG (2007) QM/MM: what have we learned, where are we, and where do we go from here? Theor Chem Acc 117 (2): 185-199. doi: 10.1007/s00214-006-0143-z [27] Martin C, Zhang Y (2005) The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 6 (11): 838-49. doi: 10.1038/nrm1761 [28] Nishioka K, Rice JC, Sarma K, Erdjument-Bromage H, Werner J, Wang YM, Chuikov S, Valenzuela P, Tempst P, Steward R, Lis JT, Allis CD, Reinberg D (2002) PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Mol Cell 9 (6): 12011213. doi: 10.1016/s1097-2765(02)00548-8 [29] Rust HL, Zurita-Lopez CI, Clarke S, Thompson PR (2011) Mechanistic studies on transcriptional coactivator protein arginine methyltransferase 1. Biochemistry 50 (16): 3332-45. doi: 10.1021/bi102022e [30] Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y (2002) G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Gene Dev 16 (14): 1779-1791. doi: 10.1101/gad989402 [31] Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, Sakihama T, Kodama T, Hamakubo T, Shinkai Y (2005) Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Gene Dev 19 (7): 815-826. doi: 10.1101/gad.1284005 [32] Tamaru H, Zhang X, McMillen D, Singh PB, Nakayama J, Grewal SI, Allis CD, Cheng XD, Selker EU (2003) Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat Genet 34 (1): 75-79. doi: 10.1038/ng1143
10 [33] Taverna SD, Li H, Ruthenburg AJ, Allis CD, Patel DJ (2007) How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14 (11): 1025-40. doi: 10.1038/nsmb1338 [34] Trievel RC (2004) Structure and function of histone methyltransferases. Crit Rev Eukaryot Gene Expr 14 (3): 147-69 [35] Wang SL, Hu P, Zhang YK (2007) Ab initio quantum mechanical/molecular mechanical molecular dynamics simulation of enzyme catalysis: The case of histone lysine methyltransferase SET7/9. J Phys Chem B 111 (14): 3758-3764. doi: 10.1021/jp067147i [36] Warshel A, Levitt M (1976) Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. J Mol Biol 103 (2): 227-49 [37] Wu H, Min J, Lunin VV, Antoshenko T, Dombrovski L, Zeng H, Allali-Hassani A, CampagnaSlater V, Vedadi M, Arrowsmith CH, Plotnikov AN, Schapira M (2010) Structural biology of human H3K9 methyltransferases. PLoS One 5 (1): e8570. doi: 10.1371/journal.pone.0008570 [38] Wysocka J, Allis CD, Coonrod S (2006) Histone arginine methylation and its dynamic regulation. Front Biosci 11: 344-55 [39] Xiao B, Jing C, Wilson JR, Walker PA, Vasisht N, Kelly G, Howell S, Taylor IA, Blackburn GM, Gamblin SJ (2003) Structure and catalytic mechanism of the human histone methyltransferase SET7/9. Nature 421 (6923): 652-656. doi: http://www. nature.com/nature/journal/v421/n6923/suppinfo/ nature01378 S1.html [40] Xiao B, Wilson JR, Gamblin SJ (2003) SET domains and histone methylation. Curr Opin Struct Biol 13 (6): 699-705 [41] Xu Q, Chu YZ, Guo HB, Smith JC, Guo H (2009) Energy Triplets for Writing Epigenetic Marks: Insights from QM/MM Free-Energy Simulations of Protein Lysine Methyltransferases. Chem-Eur J 15 (46): 1259612599. doi: 10.1002/chem.200902297 [42] Xu Q, Guo H-B, Wlodawer A, Nakayama T, Guo H (2007) The QM/MM molecular dynamics and free energy simulations of the acylation reaction catalyzed
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QM/MM MD and Free Energy Simulation Study of Methyl Transfer Processes Catalyzed by PKMTs and PRMTs 1
2
Yuzhuo Chu1∗ , Hong Guo2
(School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China) (Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996 and UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6164)
Received 16 July 2014/ Revised 19 September 2014/ Accepted 17 October 2014
Abstract: Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here we review the results of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes. Key words: methyl transfer, PKMT, PRMT, product specificity, QM/MM MD, free energy simulations, reaction mechanism.
1 Overview Methyl transfer processes play important roles in many different biological functions. One type of methyl transfer processes, histone lysine methylations occurring at the histone tails govern many important biological events, including heterochromatin formation, Xchromosome inactivation, transcriptional silencing and activation (Jenuwein 2006; Martin and Zhang 2005). These methylations are catalyzed mostly by the SET domain protein lysine methyltransferases (PKMTs) through transferring methyl groups from S-adenosylL-methionine (AdoMet) to the amino group of a lysine residue on the histone, leading to a methylated lysine residue and the cofactor byproduct S-adenosylL-homocysteine (AdoHcy) (Trievel 2004). A range of lysine (K) residues in different histones, including K4, K9, K27, K36 and K79 in histone H3, K20 in histone H4, etc have been found to be the methylation targets. The SET domain PKMTs may be divided into the following families, SUV39, SET1, SET2, EZ, RIZ, SMYD, SUV4-20, and also orphan members like SET7/9 and SET8. These members contain a similar SET domain and similar motifs surrounding the SET domain (Dillon et al. 2005). One important property of PKMTs is their product specificity, which refers to their dif∗
Corresponding author. E-mail: [email protected]
ferent ability to add one, two or three methyl groups to the target lysine (Cheng et al. 2005; Xiao et al. 2003). Specific methylation states are enriched in different regions of genes, recognized by different methyllysine binding proteins and lead to different subsequent biological events (Li et al. 2007; Taverna et al. 2007). Thus it is important to understand the mechanism underlying the product specificity of PKMTs. A specific Tyr/Phe switch position within the lysine binding site has been indicated to control the product specificity of PKMTs. This position is frequently occupied by a tyrosine in the mono-methyltransferases and by a phenylalanine or another hydrophobic amino acid in the diand tri-methyltransferases (Collins et al. 2005; Zhang et al. 2003). The mutations of other important activesite residues beyond the Tyr/Phe switch could also alter the product specificity of PKMTs (Del Rizzo et al. 2010; Wu et al. 2010; Zhang et al. 2003). However, the mechanism of how the Tyr/Phe switch and other activesite mutants alter the enzyme product specificity is still unclear due to the limitations of experimental studies. Another important methyl transfer process, protein arginine methylation, occurs in abundant eukaryotic proteins and modulates different cell signaling pathways, including gene transcription, RNA splicing, signal transduction, cell growth and proliferation (Bedford and Clarke 2009). These methyl transfer processes are catalyzed by the family of protein arginine methyltransferases (PRMTs) through the transfer of methyl
2
groups from AdoMet to the guanidine group of arginine residues in protein substrate (Lee et al. 1977). There are two major types of PRMTs which catalyze the formation of different products. Both types of enzymes catalyze the formation of ω-N G -monomethylarginine (MMA). Type I PRMTs catalyze the formation of asymmetric ω-N G , N G -dimethylarginine (ADMA), while type II PRMTs catalyze the formation of symmetric ω-NG , N G -dimethylarginine (SDMA) (Di Lorenzo and Bedford 2011). Different dimethylated product (ADMA or SDMA) might lead to different biological consequences (Wysocka et al. 2006), thus it is very important to understand the basic mechanism underlying the methylation processes and the product specificity of PRMTs. Molecular dynamics (MD) simulations based on molecular mechanics (MM) force field could be performed to obtain the structural information of the reactant complex. However, the description of the electron re-distribution during covalent bond breaking and making process requires a quantum mechanics (QM) potential (Lin and Truhlar 2007). Although high-level ab initio calculations (e.g., B3LYP/6-31G** or MP2/631G**) have been widely used for simple systems, such as proton sponges (Guo and Salahub 2001), polyacetylene (Guo and Paldus 1997) and peptides with carbon hydrogen bonds (Guo et al. 2004), these approaches are still too time consuming for studying enzyme-catalyzed reactions. The hybrid QM/MM MD simulations combine the accuracy of QM with the low computational cost of MM by treating only the reacting part of the systems quantum mechanically and the other part molecular mechanically (Warshel and Levitt 1976). These approaches have been utilized to study enzyme reactions in different biological systems (Guo et al. 2006; Guo et al. 2005; Hu et al. 2008; Hu et al. 2008; Wang et al. 2007; Xu et al. 2007; Xu et al. 2007; Xu et al. 2006; Xu et al. 2010; Yao et al. 2011; Zhang and Bruice 2008). The energetic results of SCC-DFTB and high-level B3LYP/6-31G** methods for the methyl transfer in a simple model system that resembles the methyl transfer in PKMTs and PRMTs were compared to understand the performance of the semi-empirical method in these reactions (Chu et al. 2013; Guo and Guo 2007; Xu et al. 2009). For PKMTs, although the SCC-DFTB optimized geometries along the reaction pathway seemed to be rather close to those from B3LYP/6-31G**, there are some systematic deviations of the SCC-DFTB method in the description of the energetics of the methyl transfer. To correct the errors due to the deficiency of SCC-DFTB method, the empirical correction (Guo and Guo 2007; Xu et al. 2009) was applied to the free energy curves obtained from the potential of mean force simulations. The energy curves from the corrected SCC-DFTB and B3LYP/6-31G** were very close, supporting the use of the approach
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
with the empirical correction. For PRMTs, the potential energy function obtained from SCC-DFTB is quite similar to that obtained from the B3LYP/6-31G** calculations. Therefore, no empirical correction seems to be necessary for this reaction (Chu et al. 2013). In this review, we will discuss the applications of QM/MM MD and free energy simulations in the study of methyl transfer processes catalyzed by PKMTs and PRMTs (Chu et al. 2013; Chu et al. 2010; Chu et al. 2012; Guo and Guo 2007; Xu et al. 2009). The reaction mechanism and the mechanism underlying the special properties of these enzymes, such as the product specificity, are investigated in these computational studies.
2 QM/MM MD and free energy simulations of the methyl transfers catalyzed by PKMTs 2.1
SET7/9
Initially identified as a histone3 lysine 4 (H3-K4) specific methyltransferase, SET7/9 could also regulate the functions of non-histone substrates through site-specific methylation (Chuikov et al. 2004). The Tyr/Phe switch mutant Y305F alters the enzyme from a monomethyltransferase to a di-methyltransferase. Another active-site mutant, Y245A alters the enzyme to a trimethyltransferase with weaker activity (Del Rizzo et al. 2010). In the crystal structure of SET7/9 in complex with substrate histone peptide and cofactor Sadenosylhomocysteine (AdoHcy) (PDB ID: 1O9S), the substrate nitrogen is aligned with the active methyl group of AdoMet via hydrogen bonding to the hydroxyl group of Y245 and the oxygen atom of an active-site water molecule. This water molecule is further stabilized in the active site via hydrogen bonding to the hydroxyl group of Y305 (Xiao et al. 2003). The free energy barriers of the methyl transfer processes were calculated from QM/MM MD and free energy simulations along the reaction coordinate [R = r(CM −Sδ ) – r(CM −Nε )] (Fig. 1) (Guo and Guo 2007). The calculated free energy barrier of the second methyl transfer process in wild type (wt) SET7/9 is about 5 kcal/mol higher than that of the first methyl transfer process. The higher free energy barrier of the second
r2
H3K4
Fig. 1
Nξ
r2 rI
CM Sδ
A doMet AdoMet
θ θ
rI r2
The reaction coordinate for calculating the free energy barriers of the methyl transfer process in SET7/9 (PKMTs) is R = r(CM −Sδ ) – r(CM −Nξ ). Figure adapted from (Guo and Guo 2007).
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3
bonding to Y245 and also an active-site water molecule. However, the substrate could not be well aligned with AdoMet in the second methyl transfer of wt SET7/9. In this case, the substrate is pushed away from AdoMet in the more crowded active site after it accepts one methyl group. the substrate is pushed away from AdoMet since the active site becomes more crowded after it accepts one methyl group. As for the second methyl transfer in Y305F, there is more space around the substrate since the active-site water molecule could dissociate, leaving the substrate well aligned with AdoMet and making this methyl transfer process possible to proceed (Guo and Guo 2007).
methyl transfer process makes this process difficult to proceed than the first methyl transfer. In contrast to wt SET7/9, the calculated free energy barriers of the first and second methyl transfer processes in Y305F are similar, consistent with the fact that Y305F alters the enzyme to a di-methyltransferase (Guo and Guo 2007). The reactant complex conformations for the methyl transfer processes were also obtained from QM/MM MD simulations (Fig. 2). The active-site structures of the reactant complex of the simulated methyl transfer processes indicate that for the first methyl transfer in wt SET7/9, the target nitrogen is well aligned with the active methyl group of AdoMet via hydrogen
N265 N265
Y245
o1
H3K4me
o1 2.3 2.9 h1 h2 3.6 2.6
1.8
h 4.0
Y245
Y305
1.8
Y335 W1 A295
AdoMet
Y245 AdoMet
A295
Y305 (b)
G264 h 2.2 3.2
H293
1.8 H293
Y335 W1 A295
F305
AdoMet
(c)
The active-site structures of the reactant complex for the methyl transfers in wt SET7/9 and Y305F mutant obtained from QM/MM MD simulations. (a) The first methyl transfer in wt SET7/9. (b) The second methyl transfer in wt SET7/9. (c) The second methyl transfer in Y305F. Figures adapted from (Guo and Guo 2007).
The calculated free energy barriers of the first, second and third methyl transfer processes in Y245A mutant are similar, making all these processes possible to proceed. Also, the barrier of the first methyl transfer process is about 2 kcal/mol higher than that of the first methyl transfer in wt SET7/9, consistent with the fact that the methylation catalyzed by Y245A mutant is less efficient. The reactant complex conformations of all three methyl transfer processes in Y245A indicate that the substrate nitrogen is well aligned with AdoMet. The mutation of Y245 to alanine generates more free space in the active site, making it possible for the substrate to accept three methyl groups (Yao et al. 2012). 2.2
H3K4me
o2
(a)
Fig. 2
N265
H293
o2 Y335 W1
N265
G264
N264 H3K4
N265
N265
DIM-5
DIM-5 catalyzes the tri-methylation of H3-K9 and marks chromatin regions for DNA methylation (Tamaru et al. 2003). The crystal structure of DIM5 in complex with AdoHcy and H3 peptide (PDB ID: 1PEG) indicates that the substrate lysine is stabilized in the active site via van der Waals contact between its methylene group and the aromatic side chains of F206, F281, Y283 and W318 in DIM-5 (Zhang et al. 2003). The substrate amino group also forms hydrogen bond with the hydroxyl group of another active-site residue
Y178. The mutation of the Tyr/Phe switch, F281Y, does not reduce the overall catalytic activity of DIM-5 but alters it to a mono-/di-methyltransferase (Zhang et al. 2003). QM/MM MD and free energy simulations were applied in DIM-5 to study the origin of its product specificity and its changes due to mutations (Xu et al. 2009). The calculated free energy barriers of the first, second and third methyl transfer processes in DIM-5 are similar, consistent with the fact that DIM-5 is a tri-methyltransferase. For F281Y mutant, the calculated free energy barrier of the second methyl transfer process is about 3 kcal/mol higher than the first transfer and the barrier of the third methyl transfer process is about 8 kcal/mol higher than the first transfer. The higher free energy barrier of the second and third methyl transfer processes in F281Y mutant could stop the further methylation process. These results suggest that the relative free energy barriers for the methyl transfers might be the energetic factors controlling the enzyme product specificity. The reactant complex conformations for the methyl transfer processes in DIM-5 and its F281Y mutant were also obtained from QM/MM MD simulations (Xu et al. 2009). The comparison of the reactant complex conformation of the third methyl transfer process in DIM-5 and F281Y indicates that the electron lone pair
4
Interdiscip Sci Comput Life Sci (2015) 7: 1–10 Y204
V203
Y204
V203 Y178 Y178 3.0
H3K9(me)2 4.6
1.8
1.8 Y283
H3K9(me)2 R238 W1
R238
Y283 I240
F281
Y281 W1
AdoMet AdoMet
(a)
Fig. 3
(b)
The active-site structures of the reactant complex for the third methyl transfer processes in wt DIM-5 and F281Y mutant obtained from simulations. (a) The third methyl transfer in wt DIM-5. (b) The third methyl transfer in F281Y mutant. Figures adapted from (Xu et al. 2009).
on the Nε atom of the target di-methylated lysine is well aligned with the active methyl group of AdoMet in wt DIM-5 (Fig. 3), but not in the F281Y mutant. These results suggest that the formation of reactive configuration in the reactant complex might be used as a predictor for the product specificity. Similar as the case of SET7/9, a significant difference between the active-site structure of the reactant complex in the third methyl transfer of DIM-5 and F281Y is the location of an active-site water molecule. In DIM-5, this water molecule is able to dissociate from the active site, leaving more space around the target nitrogen to form well alignment with AdoMet. As for F281Y, this water molecule is stabilized in the active site via hydrogen bonding with the hydroxyl group of Y281. The active-site space around the target nitrogen becomes more crowded and thus the target nitrogen is pushed further away from the active methyl group of AdoMet. These structural information obtained from simulations suggest that the dissociation of this active-site water molecule plays an essential role in the determination of the product specificity of DIM-5 and its Y281F mutant (Xu et al. 2009). The mutation of Y281 to tryptophan abolishes the methylation activity of DIM-5 completely (Zhang et al. 2003). Consistent with this fact, the calculated free energy barrier of the first methyl transfer of Y281W is about 6 kcal/mol higher than that of wt DIM-5. The reactant complex conformation of Y281W first methyl transfer also indicates that the target nitrogen is not well aligned with the active methyl group of AdoMet, possibly due to the steric hinder caused by the sidechain of W281, which occupies more space (Xu et al. 2009). 2.3
AdoMet I240
strate H3K20 and cofactor AdoHcy (PDB ID: 1ZKK) (Couture et al. 2005), the substrate lysine is oriented towards the cofactor via hydrogen bond interactions with the hydroxyl group of an active-site residue Tyr245 and also an active-site water molecule (W1). This water molecule is further stabilized in the active site via hydrogen bonding to Tyr334 hydroxyl group and the carbonyl oxygen of Gly-294 and Ile-297 (Fig. 4). The mutation of the Tyr/Phe switch of SET8 (Y334F) could alter this enzyme to a di-methyltransferase (Couture et al. 2005). The structural observations of Y334F mutant in complex with histone H4 bearing unmodified, mono-methylated, and di-methylated status suggest that Y334F mutant might alter the product specificity of SET8 through affecting the dissociation ability of an active-site water molecule (Couture et al. 2008). QM/MM MD and free energy simulations were applied to reveal the methyl transfer processes catalyzed by SET8 and its Y334F mutant and to study the origin of their product specificity (Chu et al. 2010). The cal-
Tyr271 K20
ADY Tyr245
Gly2944
SET8
SET8 is implicated in cell-cycle-dependent transcriptional silencing (Nishioka et al. 2002) and mitotic regulation in metazoans (Karachentsev et al. 2005). It catalyzes the specific mono-methylation of H4-K20. In the active-site structure of SET8 in complex with sub-
Tyr336 3 W1
Ile297 Tyr334
Fig. 4
The active-site structure of SET8 in complex with AdoHcy and H3K20 (PDB ID: 1ZKK) (Couture et al. 2005).
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
5
the active-site structures of the reactant complex for the first methyl transfer of SET8, the first and second methyl transfers of Y334F mutant, the lone pair of electrons on the target Nε atom is well aligned with the active methyl group of the cofactor AdoMet. However, in the active-site structures of the reactant complex for the second methyl transfer of wt SET8 and the third methyl transfer of Y334F mutant, the lone pair of electrons on the target Nε atom is not well aligned with the active methyl group of AdoMet. These results suggest that the formation of reactive configuration in the reactant complex obtained from MD simulations could be used as a predictor of the product specificity. The reactions will be less efficient if the reactive configuration could not be formed in the reactant complex, since additional energy cost will be required to generate the reactive configuration first. In the active-site structure of the reactant complex for the second methyl transfer in SET8, the active-site water molecule is stabilized via hydrogen bonds interactions with the hydroxyl group of Tyr334. The active site of the reactant complex for this methyl transfer process is too crowded, thus the target nitrogen and the active methyl group of AdoMet could not form well alignment. In the active-site structure of
culated free energy barrier of the second methyl transfer process of wt SET8 is about 6.5 kcal/mol higher than that of its first methyl transfer process. As for the Y334F mutant, the free energy barriers of the first and second methyl transfer processes are similar with that of the first methyl transfer in wt SET8. However the free energy barrier of its third methyl transfer process is about 9 kcal/mol higher than the first methyl transfer process in wt SET8. The calculated free energy barriers of the methyl transfer processes in wt SET8 and its Y334F mutant are consistent with their product specificity, which suggest that the high free energy barrier of certain methyl transfer process (for instance, the second methyl transfer in wt SET8 and the third methyl transfer in Y334F mutant) could stop the further methylation. Y334F mutant could alter the enzyme to a di-methyltransferase since it lowers the free energy barrier of the second methyl transfer process (Chu et al. 2010). The active-site structures of the reactant complex for the methyl transfers in wt SET8 and Y334F mutant obtained from the simulations indicate that their product specificity are already reflected from their reactant complex conformations (Chu et al. 2010) (Fig. 5). In Tyr271
Tyr271 Tyr245
Tyr245
3.0
2.4 3.0 1.8 H4K20 3.2
1.8
4.5 H4K20
5.0 5.0
W1
W1 AdoMet
Tyr334
AdoMet
Tyr334 (a)
(b)
Tyr271
Tyr271
Tyr271
Tyr245 Tyr245 2.2 3.0
2.9 1.8 3.0 H4K20 2.7 5.0 W1 Phe334
1.8
4.6
H4K20
1.8
11.0 H4K20 W1 AdoMet AdoMet
Phe334 (c)
Fig. 5
Tyr245
(d)
Phe334
AdoMet (e)
The active-site structures of the reactant complex for the methyl transfers in wt SET8 and Y334F mutant obtained from simulations. (a) The first methyl transfer in wt SET8. (b) The second methyl transfer in wt SET8. (c) The first methyl transfer in Y334F. (d) The second methyl transfer in Y334F. (e) The third methyl transfer in Y334F. Figures adapted from (Chu et al. 2010).
6
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
the reactant complex for the second methyl transfer in Y334F mutant, this water molecule is able to dissociate from the active site since its hydrogen bond interactions with the hydroxyl group of Tyr334 is lost due to its mutation to Phe. The dissociation of this active-site water molecule generates more space around the target nitrogen and allows it to form well alignment with the active methyl group of AdoMet. 2.4
GLP
G9a-Like-Protein (GLP) and its closely related homolog G9a are the major mammalian H3-K9 methyltransferases that target euchromatic regions and essential for murine embryogenesis (Tachibana et al. 2002). They form a heteromic complex and work cooperatively in euchromatin to exert H3-K9 methyltransferase function in vivo (Tachibana et al. 2005). The Tyr/Phe switch sites of GLP and G9a are occupied by a Phe and the mutation of this site in G9a (F1152Y) alters the enzyme from a di-methyltransferase to a mono-methyltransferase. The equivalent residue in GLP is F1209. In addition to the single-residue mutant at the Tyr/Phe switch site which could alter the product specificity of G9a, the mutation of another active-site residue, Y1206F, could alter it to a
Table 1
tri-methyltransferase. The equivalent residue in GLP is Y1124 (Collins et al. 2005) (Table 1). The calculated free energy barriers of the methyl transfer processes in wt GLP, its F1209Y, and Y1124F mutants are consistent with their product specificity (Chu et al. 2012). For wt GLP, the calculated free energy barrier of the first and second methyl transfer processes are similar. Whereas the barrier of the third methyl transfer process is about 9 kcal/mol higher than that of the first methyl transfer. For the F1209Y mutant, the free energy barrier of the second methyl transfer process is increased to about 4 kcal/mol higher than that of the first methyl transfer process. The high energy barrier of this step might stop the further methylation and thus F1209Y is a mono-methyltransferase. As for the Y1124F mutant, the free energy barrier of the third methyl transfer process is also lowered to be similar with the first methyl transfer, which allows the third methylation process to proceed. These results are consistent with the conclusions from previous studies of other PKMTs that the free energy barrier of the methyl transfer process might determine the energetic origins of the product specificity of these enzymes and their mutants (Chu et al. 2012).
The product specificity of wt G9a and its active-site mutants (with the equivalent mutants of GLP listed)
wt G9a
F1152Y of G9a (F1209Y of GLP)
Y1206F of G9a (Y1124F of GLP)
Di-methyltransferase
Mono-methyltransferase
Tri-methyltransferase
As suggested from previous PKMTs studies, the product specificity of GLP and its mutants could also be reflected from the reactant complex conformations obtained from the simulations (Chu et al. 2012). In the first, second methylations of wt GLP, the first methylation of F1209Y and the first, second and third methylations of Y1124F, the lone pair of electrons on the target Nε atom is well aligned with the active methyl group of the cofactor AdoMet in the reactant complex, consistent with the fact that these methylation processes are efficient. As for the methylations which are not efficient, such as the third methylation of wt GLP, the second, third methylations of F1209Y, the target nitrogen could not form well alignment with AdoMet. The structural information of the transition-state complex for the second methylation in wt GLP obtained from the free energy simulations indicates that the active-site water molecule could dissociate from the active-site during this methylation process. This generates more space around the target nitrogen to allow it to form well alignment with AdoMet. As for the F1209Y mutant, the structural information of the transition-state complex
for the second methylation indicates that this activesite water molecule is stabilized via hydrogen bonding to the hydroxyl group of Y1209. The active-site space around the target nitrogen is too crowded thus it could not be well aligned with AdoMet. The third methylation in GLP could not proceed since the active-site space around the target nitrogen is too crowded after two methyl groups are added. Whereas the loss of the hydroxyl group due to Y1124F mutation generates more active-site space, facilitates the well alignment of the target nitrogen and AdoMet in its third methylation step (Fig. 6). The computational results of GLP, its F1209Y, and Y1124F mutants suggest that the activesite space around the target nitrogen is an important factor for the formation of reactive configurations and the methylation activity (Chu et al. 2012).
3 QM/MM MD and free energy simulations of the methyl transfer processes catalyzed by PRMT3 QM/MM MD and free energy simulations were
Interdiscip Sci Comput Life Sci (2015) 7: 1–10 Tyr1124
Tyr1142 Tyr1142 Tyr114 1442 1.88
Phe1124
4.5
H3K9me2 3K9me2
H3K9 AdoMet
Phe1209 he1209
Phe12099
(a)
Fig. 6
3.0 1.88
AdoMet (b)
The active-site structures of the reactant complex for the third methyl transfer processes in wt GLP and Y1124F mutant obtained from simulations. (a) The third methyl transfer in wt GLP. (b) The third methyl transfer in Y1124F. Figures adapted from (Chu et al. 2012).
Peptidyl-Arg θ Nη1 Nη2
Fig. 7
Sδ AdoMet
CM r(CM−Nη2)
The reaction coordinate for calculating the free energy barriers of the methyl transfer process in PRMT3 is R = r(CM − Sδ ) – r(CM − Nη ). Figure adapted from (Chu et al. 2013).
7
active-site arginine residue. Thus the positive charge of the guanidine group is redistributed towards the amino group near Glu335 and leaving the Nη2 atom to be the possible nucleophile (Chu et al. 2013; Zhang and Cheng 2003; Zhang et al. 2000). The possible general base during the catalysis has also been discussed in PRMTs (Rust et al. 2011; Zhang et al. 2000), whereas the knowledge is very limited. A His-Asp relay system has been suggested to function as the general base (Zhang et al. 2000). However, the location of the His residue (>6˚ A from the substrate guanidine group) makes it less possible to function as the general base; the mutagenesis study also did not support its role as the general base (Rust et al. 2011). QM/MM free energy simulation results suggest that the conserved Glu326 might function as the general base. The transition-state structures obtained from the simulations indicate that the proton on the attacked nitrogen has transferred to the carboxyl group of Glu326 (Fig. 9(a)) (Chu et al. 2013). 2D free energy simulation was also performed to reveal the second methylation process and to further explore the relationship between the methyl transfer and proton transfer processes (Fig. 9(b)). The simulation results suggest that the proton on the Nη2 atom is transferred to the carboxyl group of Glu326 near the transition state and the proton and methyl transfer processes are somehow concerted (Chu et al. 2013).
4 Summary performed on one Type I PRMT, PRMT3, to study its reaction mechanism and also the origin of its product specificity which leads to the formation of ADMA product instead of the SDMA product. The calculated free energy barriers (along the reaction coordinate [Rx = r(CM − Sδ ) – r(CM − Nη )]) indicate that for both the first and second methyl transfer processes, the barriers of the methylation on the Nη1 atom of the target arginine are much higher than that of the Nη2 atom (about 7-8 kcal/mol higher). This suggests that both methyl groups would be added to the Nη2 atom and the ADMA product would be generated (Chu et al. 2013). The active-site structures of the reactant complex for the first and second methylation processes obtained from simulations also indicate that the Nη2 atom is well aligned with the active methyl group of AdoMet (Fig. 8) (Chu et al. 2013). The substrate arginine is stabilized in the active site via several active-site interactions, for instance, hydrogen bonding to the conserved “double E” residues (Glu326 and Glu335). The chargecharge interactions between Glu326 and AdoMet also helps to direct the active methyl group towards the Nη2 atom. The negative charge of Glu326 is greatly reduced through interactions with AdoMet and a nearby
QM/MM MD and free energy simulations were performed to study the catalytic mechanism and product specificity of several PKMTs and PRMTs. The computational results suggest that the dissociation ability of an active-site water molecule determines the product specificity of the Tyr/Phe switch of PKMTs. The active-site space around the target nitrogen also controls the product specificity of other PKMT mutants. The calculated free energy barriers of the methyl transfer processes are consistent with their efficiency, suggesting that these barriers might be the energetic control of the product specificity. The formation of reactive configuration in the reactant complex obtained from the simulations are also consistent with the reaction efficiency, suggesting that they could be used as a predictor for the methylation activity. As for the PRMTs, the calculated free energy barriers of the methyl transfer processes suggest that both methyl groups would be added to the Nη2 atom, thus generating the ADMA product. The reactant complex conformations also suggest that for both methyl transfer processes, the Nη2 atom is well aligned with the active methyl group of AdoMet. The computational results also suggest that one of the “double E” residues, Glu326, might function as the general base of the re-
8
Interdiscip Sci Comput Life Sci (2015) 7: 1–10
Sub
Sub R236
2.5±0.2
OE1 3.1±0.3 HH21 Nη2 E326 3.2±0.2
Nη1
3.6±0.1
HH22 2.1±0.3
1.7±0.1
2.0±0.2
2.5±0.4
R236
E335
HH22 1.7±0.1
1.9±0.3
OE1
3.2±0.2
1.7±0.1
Nη1 1.9±0.4 11.7°±6.5
Y221
37.4°±6.6
Y221
1.7±0.1 2.1±0.3
(a)
E326 ADM (b)
1.9±0.3
HH21 Nη2 R236
1.9±0.1 HH22 1.8±0.2
E335
1.8±0.2
Nη1 Y221 2.2±0.04 1.8±0.2
OE1 1.8±0.2 E326 2.0±0.3
2.0±0.04
ADM (a)
Ry=r(HH22...Nη2)−r(HH22...OE1) (Å)
The active-site structures of the reactant complex for the methyl transfers in PRMT3 obtained from simulations. (a) The first methyl transfer. (b) The second methyl transfer. Figures adapted from (Chu et al. 2013).
Sub
Fig. 9
E335
Nη2
ADM
Fig. 8
1.9±0.3
1.4
0.7
0
−1
0 1 Rx=r(CM...Sδ)−r(CM...Nη2) (Å) (b)
(a) The active-site structure of the transition state complex for the first methyl transfer in PRMT3 obtained from simulations. (b) 2D free energy contours for the methyl transfer from AdoMet to methyl arginine and the proton transfer between methyl arginine and Glu326. Figures adapted from (Chu et al. 2013).
action. The methyl transfer and proton transfer processes are somehow concerted as suggested from the 2D free energy simulations. In future studies, QM/MM MD and free energy simulation approaches could be applied to study the properties of other enzyme-catalyzed methyl transfer processes and also other important biological reactions.
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