Mechanisms of Deamidation of Asparagine Residues and Effects Of

International Journal of Molecular Sciences

Article Mechanisms of Deamidation of Asparagine Residues and Eﬀects of Main-Chain Conformation on Activation Energy

Koichi Kato 1,2,*, Tomoki Nakayoshi 2, Eiji Kurimoto 2 and Akifumi Oda 2,3 1 College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japan 2 Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan; [email protected] (T.N.); [email protected] (E.K.); [email protected] (A.O.) 3 Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan * Correspondence: [email protected]; Tel.: +81-527-980-180

Received: 31 August 2020; Accepted: 22 September 2020; Published: 24 September 2020

Abstract: Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

Keywords: deamidation; molecular dynamics simulation; quantum chemical calculation; post-translational modiﬁcation; age-related diseases

1. Introduction Deamidation of asparagine (Asn) residue is a post-translational modification observed in various proteins and has been reported to spontaneously and nonenzymatically occur in vivo and in vitro under physiological conditions [1–6]. This reaction typically proceeds through the cyclic succinimide intermediate, resulting in the production of Asp and biologically uncommon isoAsp residues in an approximately 1:3 molar ratio (Figure1)[ 7]. These conversions from Asn residues to acidic residues by deamidation affect the structural stability of proteins and are seriously disruptive to biologically important functions [5,8–12]. Some of the deamidated proteins are targeted for degradation by the ubiquitin–proteasome system [13]. Therefore, deamidation is hypothesized to serve as a molecular clock for biological processes such as protein turnover. In addition, because deamidation can result in protein denaturation and aggregation, it is suggested to trigger several age-related diseases [14–18]. In fact, deamidated residues are frequently detected in crystallins isolated from the eye lens of patients suffering from age-related nuclear cataracts [16–18]. Furthermore, isoAsp formation in vivo is involved in autoimmune diseases [19–22]. An isoAsp-containing peptide triggers an autoimmune response to the native peptide containing the original Asn residue. For instance, isoAsp formation in histone H2B has been suggested to be involved in systemic lupus erythematosus [21,22]. Furthermore, Asn deamidation

Int. J. Mol. Sci. 2020, 21, 7035; doi:10.3390/ijms21197035 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2020, 21, 7035 2 of 14 response to the native peptide containing the original Asn residue. For instance, isoAsp formation in histone H2B has been suggested to be involved in systemic lupus erythematosus [21,22]. Further- playsmore, a Asn key roledeamidation in quality plays control a key of antibody role in quality drugs. control Deamidations of antibody have drugs. been reported Deamidations to occur have in complementarity-determiningbeen reported to occur in complementarity-determ regions (CDRs) and Fcining regions regions of recombinant (CDRs) and monoclonal Fc regions of antibodies recombi- andnant human monoclonal endogenous antibodies IgG andin vivohuman[23 endogenous–27]. In particular, IgG in vivo antigen-binding [23–27]. In particular, aﬃnities antigen-bind- are reduced bying Asn affinities deamidation are reduced in CDRs. by Asn Therefore, deamidation Asn in deamidation CDRs. Therefore, is an Asn important deamidation reaction is foran important not only pathologicalreaction for analysisnot only butpathological also development analysis but of peptide also development/protein-based of pept drugs.ide/protein-based drugs.

+H2O Asp −H2O −NH3

+H O Asn succinimide 2 −H2O

isoAsp

Figure 1. Succinimide-mediated deamidation pathways of asparagine (Asn) residues. Figure 1. Succinimide-mediated deamidation pathways of asparagine (Asn) residues. Asn deamidation rate is affected by various factors, especially adjacent residues on the C-terminal Asn deamidation rate is affected by various factors, especially adjacent residues on the C-termi- side ((N + 1) residue) [28–30]. For instance, the deamidation half-time for the Asn–Gly sequence is nal side ((N+1) residue) [28–30]. For instance, the deamidation half-time for the Asn–Gly sequence is approximately 300-fold smaller than that for the Asn–Ile sequence in a pentapeptide [29]. In contrast, approximately 300-fold smaller than that for the Asn–Ile sequence in a pentapeptide [29]. In contrast, the half-time for the Asn–His sequence is approximately 9-fold longer than that for the Asn–Gly the half-time for the Asn–His sequence is approximately 9-fold longer than that for the Asn–Gly se- sequence. In the Asn–Pro sequence, for which succinimide cannot be formed, the half-time is 7000-fold quence. In the Asn–Pro sequence, for which succinimide cannot be formed, the half-time is 7000-fold longer than that for the Asn–Gly sequence in a pentapeptide. Other important factors reported to affect longer than that for the Asn–Gly sequence in a pentapeptide. Other important factors reported to Asn-deamidation rates are secondary structure, tertiary structure, quaternary structure, hydrogen-bond affect Asn-deamidation rates are secondary structure, tertiary structure, quaternary structure, hydro- formation, surface exposure, and solvent molecules [5,7,10,31,32]. Based on this information, gen-bond formation, surface exposure, and solvent molecules [5,7,10,31,32]. Based on this infor- some predictive methods for Asn-deamidation rates in proteins have been proposed [33–38]. mation, some predictive methods for Asn-deamidation rates in proteins have been proposed [33–38]. A predictive method produced by Robinson and Robinson is currently considered to be the most reliable A predictive method produced by Robinson and Robinson is currently considered to be the most method [33,34]. In this method, amino acid sequence, secondary structures, and hydrogen bonds reliable method [33,34]. In this method, amino acid sequence, secondary structures, and hydrogen with other residues are used for the prediction. These factors are involved in succinimide formation bonds with other residues are used for the prediction. These factors are involved in succinimide for- from Asn residues. Succinimide formation is considered to be caused by nucleophilic attack of the mation from Asn residues. Succinimide formation is considered to be caused by nucleophilic attack amide nitrogen of the (N + 1) residue to the amide carbon of the Asn side chain, and proceeds via the of the amide nitrogen of the (N+1) residue to the amide carbon of the Asn side chain, and proceeds tetrahedral intermediate that includes gem-hydroxylamine (Figure2). Structural parameters involved via the tetrahedral intermediate that includes gem-hydroxylamine (Figure 2). Structural parameters in the above nucleophilic attack are expected to be important factors for deamidation rates [35–38]. involved in the above nucleophilic attack are expected to be important factors for deamidation rates The distance between the main-chain nitrogen of the (N + 1) residue and amide carbon of the Asn [35–38]. The distance between the main-chain nitrogen of the (N+1) residue and amide carbon of the side chain (NN+1–Cγ distance) in crystal structures is used to predict deamidation rate in monoclonal Asn side chain (NN+1–Cγ distance) in crystal structures is used to predict deamidation rate in mono- antibodies [35,37,38]. Furthermore, these methods use dihedral angles of Asn residues in crystal clonal antibodies [35,37,38]. Furthermore, these methods use dihedral angles of Asn residues in crys- structures as a parameter for predicting deamidation rates. In cyclic peptides containing the Asn–Gly tal structures as a parameter for predicting deamidation rates. In cyclic peptides containing the Asn– sequence, the deamidation rate is higher in the peptide with the shorter NN+1–Cγ distance [39,40]. Gly sequence, the deamidation rate is higher in the peptide with the shorter NN+1–Cγ distance [39,40]. In addition, deamidation is facilitated when the NN+1–Cγ–Oγ angle is <128◦. However, deamidation In addition, deamidation is facilitated when the NN+1–Cγ–Oγ angle is <128°. However, deamidation rate is low in a cyclic peptide satisfying these parameters (Lys–Asn–Gly–Arg–Glu–NH2). Therefore, rate is low in a cyclic peptide satisfying these parameters (Lys–Asn–Gly–Arg–Glu–NH2). Therefore, although these parameters are key in determining deamidation rate, other factors are also important. although these parameters are key in determining deamidation rate, other factors are also important. Those parameters might be difficult to apply to the flexible side chain of Asn residues in protein Those parameters might be difficult to apply to the flexible side chain of Asn residues in protein structures. Therefore, the relevance for deamidation rate of the NN+1–Cγ distance and dihedral angles structures. Therefore, the relevance for deamidation rate of the NN+1–Cγ distance and dihedral angles in crystal structures is unclear for calculations of protein dynamics. in crystal structures is unclear for calculations of protein dynamics.

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−NH3

Asn gem-hydroxylamine succinimide

cyclization deammoniation

Figure 2. Proposed two-step succinimide formation pathway of Asn residues. Figure 2. Proposed two-step succinimide formation pathway of Asn residues. Some computational studies have reported the succinimide formation mechanism of Asn and the Some computational studies have reported the succinimide formation mechanism of Asn and changes of dihedral angles with the deamidation reaction using quantum chemical calculations [41–44]. the changes of dihedral angles with the deamidation reaction using quantum chemical calculations The structural changes with succinimide formation from Asn are relatively small (change of N-Cα-C-N [41–44]. The structural changes with succinimide formation from Asn are relatively small (change of and N-Cα-Cβ-Cγ: 35–50◦, C-N-Cα-C: nearly constant) in comparison with other nonenzymatic N‒Cα‒C‒N and N‒Cα‒Cβ‒Cγ: 35–50°, C‒N‒Cα‒C: nearly constant) in comparison with other nonen- modifications in some previous studies (the maximum change of dihedral angles: 214 )[45–49]. zymatic modifications in some previous studies (the maximum change of dihedral angles:◦ 214°) [45– Therefore, the flexibilities of the main chain are assumed not to be a primary factor in determining 49]. Therefore, the flexibilities of the main chain are assumed not to be a primary factor in determining deamidation rate. However, previous computational studies of nonenzymatic modification of amino deamidation rate. However, previous computational studies of nonenzymatic modification of amino acids have not considered protein structure. In this study, conformations of Asn residues in a acids have not considered protein structure. In this study, conformations of Asn residues in a protein protein were first investigated to analyze the influence of three-dimensional (3D) structure on the Asn were first investigated to analyze the influence of three-dimensional (3D) structure on the Asn de- deamidation rate. We focused on the 3D structure of γS-crystallin, which is targeted for deamidation amidation rate. We focused on the 3D structure of γS-crystallin, which is targeted for deamidation analysis in some previous studies [32,50–52]. The experimental structure registered in the protein data analysis in some previous studies [32,50–52]. The experimental structure registered in the protein bank (PDB) was obtained, and Asn conformations in γS-crystallin were investigated. Furthermore, data bank (PDB) was obtained, and Asn conformations in γS-crystallin were investigated. Further- molecular dynamics (MD) simulations were performed to evaluate conformational flexibilities of Asn more, molecular dynamics (MD) simulations were performed to evaluate conformational flexibilities residues. Second, the reaction mechanisms and activation energy barriers in succinimide formation of Asn residues. Second, the reaction mechanisms and activation energy barriers in succinimide for- from Asn residues were investigated using quantum chemical calculations. Two different initial mation from Asn residues were investigated using quantum chemical calculations. Two different structures were constructed based on the Asn conformations in the experimental and calculated initial structures were constructed based on the Asn conformations in the experimental and calcu- structures. As the deamidation rate in the phosphate buffer is higher than that in Tris–HCl buffer [34], lated structures. As the deamidation rate in the phosphate buffer is higher than that in Tris–HCl a dihydrogen phosphate ion was employed as the catalyst. To compare mechanisms and activation buffer [34], a dihydrogen phosphate ion was employed as the catalyst. To compare mechanisms and energy barriers, water-catalyzed and carbonate-catalyzed reactions were also analyzed. activation energy barriers, water-catalyzed and carbonate-catalyzed reactions were also analyzed. 2. Results and Discussion 2. Results and Discussion 2.1. Structural Features of Frequently Deamidated Asn Residues 2.1. Structural Features of Frequently Deamidated Asn Residues To investigate the effects of Asn conformation on deamidation rate, all Asn residues in the γS-crystallinTo investigate structure the were effects examined of Asn (Figure conformation3). The level on ofdeamidation deamidation rate, at Asn14, all Asn Asn53 residues and Asn143in the γS- iscrystallin high [32]. structure Notably, were approximately examined 60%(Figure of Asn143). The is level reported of deamidation to be deamidated at Asn14, in Asn53 water-insoluble and Asn143 γS-crystallinis high [32]. extracted Notably, from approximately eye lenses of 60% patients of Asn14 with age-relatedis reported nuclear to be deamidated cataracts. Namely, in water-insoluble these Asn residuesγS-crystallin are frequently extracted deamidated from eye residues.lenses of Inpatients contrast, with Asn37 age-related and Asn76 nuclear are infrequently cataracts. Namely, deamidated: these theirAsn deamidation residues are ratios frequently are 15% deamidated and 8%, respectively. residues. In Dihedral contrast, angles Asn37 of and all AsnAsn76 residues are infrequently (Figure4) in de- γS-crystallinamidated: their are shown deamidation in Table ratios1. The are dihedral 15% and angles 8%, respectively. for H–N–C α Dihedral–H, defined angles as ϕ ofH ,all of Asn frequently residues deamidated(Figure 4) in residues γS-crystallin were are13.2 shown, 19.4 inand Table 10.4 1. ,The respectively. dihedral angles They arefor H–N–C thereforeα–H, syn defined periplanar. as φH, − ◦ ◦ ◦ Onof the frequently other hand, deamidated Asn37 was residues anti periplanar, were −13.2°, and 19.4° Asn76 and was10.4°, neither. respectively. Here, theThey conformations are therefore ofsyn Asn14,periplanar. Asn53 On and the Asn143 other werehand, defined Asn37 aswas syn anti conformations, periplanar, and and Asn76 that of was Asn37 neither. was Here, defined the as confor- anti conformation.mations of Asn14, Asn residues Asn53 and with Asn143 syn conformations were defined are as considered syn conformations, to have extensive and that deamidation. of Asn37 was Thedefined main-chain as anti conformations conformation. of Asn Asn residues residues with may sy ben importantconformations for the are deamidation considered rate.to have To analyzeextensive proteindeamidation. dynamics, The a main-chain 2000-ns MD conformations simulation was of performedAsn residues for may the γ beS-crystallin important structure. for the deamidation The root meanrate. square To analyze deviation protein (RMSD) dynamics, plot is shown a 2000- inns Figure MD 5simulation. The RMSD was value performed at the end for of the simulationγS-crystallin wasstructure. approximately The root 3.0 mean Å, and square a large deviation structural (RMSD) change plot was is shown not observed. in Figure The 5. dihedralThe RMSD angles value of at all the Asnend residues of the simulation in the final was structure approximately of the simulation 3.0 Å, and are a shownlarge structural in Table2 .change Asn14 was and Asn143not observed. formed The syndihedral conformations, angles of and all Asn Asn37 residues and Asn76 in the formed final stru anticture conformations of the simulation in the are final shown structure. in Table Although 2. Asn14 Asn53and Asn143 was not formed a syn conformation syn conformations, in the final and structure,Asn37 and all Asn76 frequently formed deamidated anti conformations Asn residues in the were final synstructure. conformations Although for Asn53 most of was the not final a 10syn ns conformati of simulationon in (Table the final3). Asn37 structure, and Asn76all frequently formed deami- anti dated Asn residues were syn conformations for most of the final 10 ns of simulation (Table 3). Asn37 and Asn76 formed anti conformations throughout the simulation. Dihedral angles φ in the frequently

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Int.Int. J. Mol. Sci. Sci. 2020,, 21,, x 7035 FOR PEER REVIEW 4 of 14 Int.deamidated J. Mol. Sci. 2020 residues, 21, x FOR werePEER REVIEWabout 55° throughout the simulation (Figure S1). The values of χ were4 of 14 not constant through the last 10 ns of the trajectories for all Asn residues. The dihedral angles ψ of fre- deamidated residues were about 55° throughout the simulation (Figure S1). The values of χ were not quently deamidated residues were about 40°. Those of the infrequently deamidated residues Asn37 deamidatedconformationsconstant through residues throughout the were last about 10 the ns 55° simulation. of throughoutthe trajecto Dihedralries the forsimulation angles all Asnϕ (Figurresidues.in thee frequently S1). The The dihedral values deamidated ofangles χ were residuesψ ofnot fre- constantand Asn76 through were the about last 20°10 nsand of 150°, the trajecto respectively.ries for Therefore, all Asn residues. dihedral The angles dihedral φ, ψ, andangles φH ψwere of fre- com- werequently about deamidated 55◦ throughout residues the were simulation about 40°. (Figure Those S1). of the The infrequently values of χ deamidatedwere not constant residues through Asn37 quentlymon indeamidated frequently residuesdeamidated were residues; about 40°. however, Those ofonly the φ infrequentlyH was common deamidated in infrequently residues deamidated Asn37 theand last Asn76 10 ns were of the about trajectories 20° and for150°, all respectively. Asn residues. Therefore, The dihedral dihedral angles anglesψ of φ frequently, ψ, and φH deamidated were com- andresidues. Asn76 were about 20° and 150°, respectively. Therefore, dihedral angles φ, ψ, and φH were com- residuesmon in frequently were about deamidated 40◦. Those residues; of the infrequentlyhowever, only deamidated φH was common residues in infrequently Asn37 and Asn76deamidated were mon in frequently deamidated residues; however, only φH was common in infrequently deamidated aboutresidues. 20◦ and 150◦, respectively. Therefore, dihedral angles ϕ, ψ, and ϕH were common in frequently residues. deamidated residues; however, only ϕH was common in infrequently deamidated residues.

Figure 3. Conformation of Asn residues in γS-crystallin. (A) Experimentally determined structure of γS-crystallin. (B–E) Conformation of Asn residues in γS-crystallin. Figure 3. Conformation of Asn residues in γS-crystallin. (A) Experimentally determined structure of FigureFigureγS-crystallin. 3. Conformation 3. Conformation (B–E) Conformation of Asn of Asn residues residues of Asn in inγ S-crystallin.residuesγS-crystallin. in γ (S-crystallin.A) ( AExperimentally) Experimentally determined determined structure structure of of γS-crystallin.γS-crystallin. (B– (EB)– ConformationF) ConformationAB of Asn of Asn residues residues in inγS-crystallin.γS-crystallin. ABχ φ CH2CONH2 ABφ ψ H χ N CHC 2CONH2 χ φH φ ψ φ CH2CONH2 φ ψ HN CH N C H H H H Figure 4. Dihedral angles used in this study to analyze the main-chain conformationof Asn residues (A for Figure 4. Dihedral angles used in this study to analyze the main-chain conformationof Asn residues φ, ψ, and χ, B for φH). Figure(A 4. for Dihedralϕ, ψ, and anglesχ, B forusedϕ Hin). this study to analyze the main-chain conformationof Asn residues (A for Figure 4. Dihedral angles used in this study to analyze the main-chain conformationof Asn residues (A for φ, ψ, and χ, B for φH). φ, ψ, and χ, B for φH).

Figure 5. Root mean square deviation (RMSD) plots for the main-chain atoms of γS-crystallin. Figure 5. Root mean square deviation (RMSD) plots for the main-chain atoms of γS-crystallin.

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Int. J. Mol. Sci. 2020, 21, 7035 5 of 14 Table 1. Dihedral angles (degrees) of Asn residues in the experimentally determined structure.

Dihedral Angle/Degree Table 1. Dihedral angles (degrees) of Asn residues in the experimentally determined structure. φ Residue φ ψ χ H Asn14 49.8Dihedral 26.1 Angle−41.9/Degree −13.2 ResidueAsn37 −125ϕ ψ155 − χ68.8 ϕ173H Asn53Asn14 49.879.8 26.1−4.29 41.9−59.9 13.219.4 − − Asn76Asn37 107125 155160 68.8−166 17343.6 − − Asn143Asn53 79.870.9 4.2919.5 59.9−54.4 19.410.4 − − Asn76 107 160 166 43.6 − Asn143 70.9 19.5 54.4 10.4 Table 2. Dihedral angles (degrees) of Asn residues in the− final structure of the molecular dynamics (MD) simulation. Table 2. Dihedral angles (degrees) of Asn residues in the ﬁnal structure of the molecular dynamics (MD) simulation. Dihedral Angle/Degree φ Residue φ ψ χ H Dihedral Angle/Degree Asn14 65.49 31.47 −52.05 −7.31 Residue Asn37 −76.63ϕ − ψ17.67 χ−79.28 ϕ−H154.86 Asn53Asn14 65.4946.48 31.4744.84 52.05−171.13 7.31−38.94 − − Asn37 76.63 17.67 79.28 154.86 Asn76 −−152.31 − 154.04 − 55.14 − 155.48 Asn53 46.48 44.84 171.13 38.94 Asn143 59.88 43.14 − −43.95 − −13.20 Asn76 152.31 154.04 55.14 155.48 − Asn143 59.88 43.14 43.95 13.20 Table 3. Percent of syn/anti conformation for the− last 10 ns of− the MD simulation.

Table 3. Percent of Asn14 syn/anti conformationAsn37 Asn53 for the lastAsn76 10 ns of theAsn143 MD simulation.

syn 95.8Asn14 Asn370 Asn5396.9 Asn760 Asn143 99.1 syn 95.8 0 96.9 0 99.1 anti 0 50.4 0 70.8 0 anti 0 50.4 0 70.8 0

To evaluate the influence of backbone flexibility on the deamidation rate, the root mean square To evaluate the influence of backbone flexibility on the deamidation rate, the root mean square fluctuation (RMSF) values of the final 10 ns trajectories were calculated (Figure 6A). As the highest fluctuation (RMSF) values of the final 10 ns trajectories were calculated (Figure6A). As the highest RMSF value was 1.60 Å at Ile160, the main chain of γS-crystallin was not highly flexible. Relatively RMSF value was 1.60 Å at Ile160, the main chain of γS-crystallin was not highly flexible. Relatively flexible regions (RMSF > 1.00 Å) are indicated in the whole structure after MD simulations (Figure flexible regions (RMSF > 1.00 Å) are indicated in the whole structure after MD simulations (Figure6B). 6B). No Asn residues were located on the flexible regions. Although Asn76 was the most flexible Asn No Asn residues were located on the flexible regions. Although Asn76 was the most flexible Asn residue, it is infrequently deamidated. Therefore, the flexibility of the main chain is considered to residue, it is infrequently deamidated. Therefore, the flexibility of the main chain is considered to have have little effect on Asn deamidation. little effect on Asn deamidation.

Figure 6. Flexibility ofof thethe backbone backbone of ofγ S-crystallin.γS-crystallin. (A ()A The) The root root mean mean square square ﬂuctuation fluctuation (RMSF) (RMSF) plot plotof γ S-crystallin.of γS-crystallin. (B) The (B) ﬁnalThe structurefinal structure of γS-crystallin of γS-crystallin obtained obtained by MD by simulations. MD simulations. The segments The seg- of mentsbackbone of backbone where RMSF where values RMSF are valu>1.0es Åare are >1.0 shown Å are in shown red. in red. 2.2. Deamidation Mechanism in the Syn Conformation 2.2. Deamidation Mechanism in the Syn Conformation Toinvestigate the reaction mechanism and activation energy barrier, quantum chemical calculations To investigate the reaction mechanism and activation energy barrier, quantum chemical calcu- were performed. The model compound used in this study was Asn capped with acetyl (Ac) and lations were performed. The model compound used in this study was Asn capped with acetyl (Ac) methylamino (NMe) groups on the N- and C-termini, respectively (Figure7). Two H 2O molecules

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 14 Int. J. Mol. Sci. Sci. 20202020,, 2121,, x 7035 FOR PEER REVIEW 6 of 14 and methylamino (NMe) groups on the N- and C-termini, respectively (Figure 7). Two H2O molecules and methylamino (NMe) groups on the N- and C-termini, respectively (Figure 7). Two H2O molecules and one dihydrogen phosphate (H2PO4−) ion or one bicarbonate (HCO3−) ion was included in the and oneone dihydrogendihydrogen phosphatephosphate (H(H2POPO4−)) ion ion or or one bicarbonate (HCO(HCO3−) )ion ion was was included included in the calculations as catalysts. These molecules2 4− and ions abundantly exist in vivo3− and have been suggested calculations as catalysts. These molecules and ions abundantly exist in vivo and have been suggested to catalyze post-translational modification of proteins [41–49]. The optimized geometries of the cy- to catalyze post-translational post-translational modification modiﬁcation of of pr proteinsoteins [41–49]. [41–49 ].The The optimize optimizedd geometries geometries of the of thecy- clization step, which occur by nucleophilic attack by the (N+1) amide nitrogen to carbonyl carbon of cyclizationclization step, step, which which occur occur by by nucleophilic nucleophilic attack attack by by the the (N+1 (N +) amide1) amide nitrogen nitrogen to carbonyl to carbonyl carbon carbon of the side chain, for the syn conformation in the phosphate-catalyzed reaction are shown in Figure 8. ofthe the side side chain, chain, for for the the syn syn conformation conformation in in the the ph phosphate-catalyzedosphate-catalyzed reaction reaction are are shown shown in in Figure Figure 88.. Those of water-catalyzed and carbonate-catalyzed reactions are shown in Figures S2 and S3. In the Those of water-catalyzedwater-catalyzed and carbonate-catalyzed reactions are shown in Figures S2 and S3.S3. In the reactant complex (RC), three hydrogen bonds were formed between the Asn residue and the H2PO4− reactant complexcomplex (RC),(RC), threethree hydrogenhydrogen bondsbonds werewere formed formed between between the the Asn Asn residue residue and and the the H H2PO2PO44−− ion (Figure 8). A hydrogen bond between the amide oxygen of the (N−1) residue and the H2PO4− ion ion (Figure(Figure8 8).). A A hydrogen hydrogen bond bond between between the the amide amide oxygen oxygen of of the the ( N (N−1)1) residueresidueand and the the H HPO2PO4− ion was not involved in the cyclization reaction, while it might stabilize the− conformation of RC.2 In4 addi-− was notnot involvedinvolved inin the the cyclization cyclization reaction, reaction, while while it it might might stabilize stabilize the the conformation conformation of RC.of RC. In additionIn addition to this hydrogen bond, the amide NH hydrogen of (N+1) residue, and amide oxygen of the side totion this to hydrogenthis hydrogen bond, bond, the amide the amide NH hydrogen NH hydrogen of (N +of1) (N+1 residue,) residue, andamide and amide oxygen oxygen of the of side the chain side chain formed hydrogen bonds with the H2PO4− ion. The cyclization step was started by proton trans- formedchain formed hydrogen hydrogen bonds bonds with thewith H thePO H2POion.4− ion. The The cyclization cyclization step step was was started started by by proton proton transfer transfer between these hydrogen-bonding2 atoms.4− Proton transfer between the (N+1) amide nitrogen and betweenfer between these these hydrogen-bonding hydrogen-bonding atoms. atoms. Proton Proton transfer transfer between between the ( Nthe+ (1)N+1 amide) amide nitrogen nitrogen and and the the H2PO4− ion was almost completed in the transition state 1 (TS1). In contrast, the proton migrating Hthe2PO H24PO− ion4− ion was was almost almost completed completed in the in transitionthe transition state state 1 (TS1). 1 (TS1). In contrast, In contrast, the proton the proton migrating migrating from from the H2PO4− ion to amide oxygen of side chain was located between those atoms. Therefore, the thefrom H 2thePO H4−2POion4− to ion amide to amide oxygen oxygen of side of chain side chain was located was located between between those atoms. those atoms. Therefore, Therefore, the (N +the1) (N+1) amide hydrogen is considered to be abstracted by the H2PO4− ion early in the cyclization step, amide(N+1) amide hydrogen hydrogen is considered is considered to be abstractedto be abstracted by the by H thePO H2POion4− early ion early in the in cyclization the cyclization step, step, and and this proton abstraction is expected to enhance the nucleophilicity2 4− of amide nitrogen. Along with thisand protonthis proton abstraction abstraction is expected is expected to enhance to enhanc thee nucleophilicity the nucleophilicity of amide of amide nitrogen. nitrogen. Along Along with thosewith those proton transfers, the distance between the (N+1) amide nitrogen and the amide carbon of side protonthose proton transfers, transfers, the distance the distance between between the (N the+ 1) (N+1 amide) amide nitrogen nitrogen and the and amide the amide carbon carbon of side of chain side chain was shortened in TS1 (2.20 Å). The completion of those proton transfers and the five-membered waschain shortened was shortened in TS1 in (2.20 TS1 Å).(2.20 The Å) completion. The completion of those of those proton proton transfers transfers and theand ﬁve-membered the five-membered ring ring formation resulted in a tetrahedral intermediate 1 (INT1) formation. The nitrogen of the (N+1) formationring formation resulted resulted in a tetrahedral in a tetrahedral intermediate intermediate 1 (INT1) 1 (INT1) formation. formation. The nitrogen The nitrogen of the (N of+ the1) amide(N+1) amide formed no hydrogen bond with the H2PO4− ion. In contrast, the H2PO4− ion formed two hydro- formedamide formed no hydrogen no hydrogen bond with bond the with H thePO H2ion.PO4− Inion. contrast, In contrast, the H thePO H2POion4− ion formed formed two two hydrogen hydrogen bonds with the (N−1) amide oxygen2 and4− OH hydrogen of the gem2 -hydroxylamine4− moiety. A hy- bondsgen bonds with with the ( Nthe (1)N amide−1) amide oxygen oxygen and OHand hydrogenOH hydrogen of the ofgem the-hydroxylamine gem-hydroxylamine moiety. moiety. A hydrogen A hydrogen bond between− the (N−1) amide oxygen and the H2PO4− ion was maintained throughout the bonddrogen between bond between the (N the1) amide (N−1) oxygenamide oxygen and the and H PO the Hion2PO was4− ion maintained was maintained throughout throughout the reaction. the reaction. Proton transfer− for the cyclization step in2 the4 −water- and carbonate-catalytic reactions was Protonreaction. transfer Proton for transfer the cyclization for the cyclization step in the step water- in the and water- carbonate-catalytic and carbonate-catalytic reactions wasreactions similar was to similar to that in the phosphate-catalytic reaction (Figures S2 and S3). Although the hydrogen bond thatsimilar in theto that phosphate-catalytic in the phosphate-catalytic reaction (Figures reaction S2 (Figures and S3). S2 Although and S3). Although the hydrogen the hydrogen bond between bond between the HCO3− ion and (N−1) amide oxygen was formed in INT1, it was not observed in RC and thebetween HCO theion HCO and3− ion (N and1) ( amideN−1) amide oxygen oxygen was formed was formed in INT1, in INT1, it was it notwas observed not observed in RC in and RC TS1.and TS1. In the3− carbonate-catalyzed− reaction, stabilization of the complexes by their interaction between InTS1. the In carbonate-catalyzed the carbonate-catalyzed reaction, reaction, stabilization stabilizat of theion complexesof the complexes by their by interaction their interaction between between the Asn the Asn residue and the HCO3− ion was relatively weak. residuethe Asn andresidue the HCOand the3− ionHCO was3− ion relatively was relatively weak. weak.

Figure 7. The structure of the model compound. Figure 7. The structure of the model compound.

FigureFigure 8. 8. OptimizedOptimized geometries geometries of the of cyclization the cyclization step for step the syn for conformation the syn conformation in the phosphate- in the Figure 8. Optimized geometries of the cyclization step for the syn conformation in the phosphatephosphate-catalyzedcatalyzed reaction. The reaction. carbon, The nitrogen, carbon, oxygen, nitrogen, and oxygen, phosphorus and phosphorus atoms are illustrated atoms are in illustrated gray, blue, in catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, gray,red, and blue, orange, red, and respectively. orange, respectively. Selected in Selectedteratomic interatomic distances are distances in units are of inÅ. units of Å. red, and orange, respectively. Selected interatomic distances are in units of Å. TheThe optimizedoptimized geometriesgeometries inin thethe deammoniationdeammoniation stepstep ofof thethe phosphate-catalyzedphosphate-catalyzed reactionreaction areare shownThe in optimized Figure9. A geometries tetrahedral in intermediate the deammoniation 2 (INT2) wasstep formed of the phosphate-catalyzed by rearrangement of thereaction H PO are−− shown in Figure 9. A tetrahedral intermediate 2 (INT2) was formed by rearrangement of the H2 2PO4 4 − ion.shown The in positionFigure 9. of A the tetrahedral H PO − intermediateion and the conformation 2 (INT2) was offormed the gem by-hydroxylamine rearrangement of moiety the H2 werePO4 ion. The position of the H22PO44 ion and the conformation of the gem-hydroxylamine moiety were ion. The position of the H2PO4− ion and the conformation of the gem-hydroxylamine moiety were consideredconsidered to be easily easily changed changed because because these these ions ions are are abundant abundant under under physiological physiological conditions. conditions. In considered to be easily changed because these ions are abundant under physiological conditions. In

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− INT2,In INT2, the theH2PO H24PO ion4− formedion formed two hydrogen two hydrogen bonds bonds with the with nitrogen the nitrogen and OH and hydrogen OH hydrogen atoms atomsof the gemof the-hydroxylaminegem-hydroxylamine moiety moiety(1.77 Å (1.77and 1.63 Å and Å, respectively). 1.63 Å, respectively). The proton The transfers proton between transfers these between hydrogen-bondedthese hydrogen-bonded atoms resulted atoms in resulted TS2 formation in TS2 formation(Figure 9). In (Figure TS2, these9). In proton TS2, these transfers proton were transfers almost completed,were almost and completed, the C‒N anddistance the C-N in gem distance-hydroxylamine in gem-hydroxylamine moiety was stretched moiety was by 0.37 stretched Å. In the by 0.37prod- Å. uctIn thecomplex product (PC), complex the C‒ (PC),N distance the C-N was distance 4.26 Å. was Therefore, 4.26 Å. Therefore,the C‒N bond the C-N was bond cleaved was after cleaved comple- after tioncompletion of proton of transfer. proton transfer. Proton transfers Proton transfers in the water-catalyzed in the water-catalyzed reaction reaction are not arecompleted not completed in the TS in ofthe the TS deammoniation of the deammoniation step (Fig stepure (Figure S4). In S4). contrast, In contrast, the C the‒N C-Nbond bond was wascleaved cleaved after after completion completion of protonof proton transfer transfer in the in thecarbonate-catalyzed carbonate-catalyzed reaction reaction (Figure (Figure S5). The S5). protonatio The protonationn of the ofnitrogen the nitrogen atom ofatom gem of-hydroxylaminegem-hydroxylamine has been has suggested been suggested to occur to occurin the in INT2 the INT2[43,44], [43 and,44], its and proton its proton transfer transfer is con- is sideredconsidered to precede to precede the C–N the C–N bond bond cleavage. cleavage. Our Ourresult resultss of the of intrinsic the intrinsic reaction reaction coordinate coordinate (IRC) (IRC) cal- culationcalculation also also suggested suggested that thatthe nitrogen the nitrogen atom atom of gem of-hydroxylaminegem-hydroxylamine is protonated is protonated in the inearly the stage early − − ofstage TS2 of formation TS2 formation in the inreaction the reaction catalyzed catalyzed by water, by water, H2PO H4 2andPO 4HCO− and3 HCOions. 3− ions.

FigureFigure 9. 9. OptimizedOptimized geometries geometries of the of deammoniation the deammoniation step for stepthe syn for conformation the syn conformation in phosphate- in catalyzedphosphate-catalyzed reaction. The reaction. carbon, Thenitrogen, carbon, oxygen, nitrogen, and oxygen, phosphorus and phosphorus atoms are illustrated atoms are in illustrated gray, blue, in red,gray, and blue, orange, red, and respectively. orange, respectively. Selected interatomic Selected interatomic distances are distances in units are of Å. in units of Å.

TheThe changes of dihedraldihedral anglesangles through through the the cyclization cyclization step step in in the the phosphate-catalyzed phosphate-catalyzed reaction reaction are areshown shown in Table in Table4. There 4. There were were no signiﬁcant no significant changes changes of ϕ in of all φ steps, in all and steps,ϕ remained and φ remained around 55 around◦ from TS155° fromto PC. TS1 The to change PC. The of ψchangein conversion of ψ in fromconversion RC to TS1 from was RC the to largest TS1 was change the largest during change the reaction during (31.2 the◦). reactionThese changes (31.2°). ofThese dihedral changes angles of dihedral were relatively angles smallerwere relatively than that smaller for succinimide than that for formation succinimide from formationAsp [46,47 from,49]. SuccinimideAsp [46,47,49]. formation Succinimide from formation Asn causes from small Asn conformational causes small conformational changes in comparison changes ϕ inwith comparison its formation with from its formation Asp. As fromH was Asp. constant, As φH the was syn constant, conformation the syn was conformation maintained throughoutwas main- tainedthe cyclization throughout and the deammoniation cyclization and steps. deammoniation The changes steps. of dihedral The changes angles of indihedral the water-catalyzed angles in the water-catalyzedreaction were similar reaction to those were in similar the phosphate-catalyzed to those in the phosphate-catalyzed reaction (Table S1). reaction In contrast, (Table the S1). alteration In contrast,of the the dihedral alteration angle of ψthein dihedral the carbonate-catalyzed angle ψ in the carbonate-catalyzed reaction was relatively reaction small was in TS1 relatively formation small from in TS1RC (17.1formation◦). In thefrom PC RC of all(17.1°). reactions, In theϕ PCand ofψ allare reactions, approximately φ and 55ψ ◦areand approximately 140◦, respectively. 55° and Dihedral 140°, respectively.angles of ϕ and DihedralϕH in angles the optimized of φ and geometries φH in the optimized for the succinimide geometries formation for the succinimide were consistent formation with werethose consistent in the Asn with residues those in in the the structure Asn residues of γS-crystallin in the structure obtained of γ byS-crystallin the MD simulation. obtained by Therefore, the MD simulation.dihedral angles Therefore, of ϕ and dihedralϕH can angles be used of φ to and predict φH can the be Asn used deamidation to predict the rate. Asn On deamidation the other hand, rate. Onψ and theχ otherwere hand, diﬀerent ψ and from χ were those different of frequently fromdeamidated those of freque residuesntly deamidated in the PDB andresidues MD simulations.in the PDB andThese MD dihedral simulations. angles These are assumed dihedral to angles not be are important assumed for to deamidation not be important rate. for deamidation rate.

Table 4. 4. DihedralDihedral angles angles (degrees) (degrees) of the of theoptimized optimized geom geometriesetries for the for syn the conformation syn conformation in the inphos- the phate-catalyzedphosphate-catalyzed reaction. reaction.

DihedralDihedral Angle Angle/Degree/Degree

ϕ ψ χ ϕH φ φ ψ χ H RC 71.04 105.2 174.0 4.917 RC 71.04 −105.2 − −174.0 4.917 TS1 57.74 136.4 168.6 7.248 TS1 57.74 −136.4 168.6− −7.248 INT1 55.41 142.0 149.5 9.097 INT1 55.41 −142.0 149.5− −9.097 INT2 54.72 141.2 146.0 7.442 − − INT2 TS254.72 54.95 −141.2141.6 146.2146.0 6.697 −7.442 − − TS2 PC54.95 54.74 −141.6140.5 139.3146.2 6.901 −6.697 − − PC 54.74 −140.5 139.3 −6.901

2.3. Deamidation Mechanism in the Anti Conformation

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Int. J. Mol.For Sci. comparison 2020, 21, x FOR of thePEER Asn REVIEW deamidation mechanism between syn and anti conformation, quan-8 of 14 2.3. Deamidation Mechanism in the Anti Conformation tum chemical calculations for the anti conformation were also performed. The optimized geometries For comparison of the Asn deamidation mechanism between syn and anti conformation, quan- of theFor cyclization comparison step of in the the Asn phosphate-catalyzed deamidation mechanism reaction between are shown syn and in anti Figu conformation,re 10. Overall, quantum proton tumtransferchemical chemical and calculations nucleophilic calculations for the attackfor anti the for conformationanti cyclization conformation werewere werealso similarperformed. also to performed. those Thein the optimized The syn optimized conformation. geometries geometries In of ad- the of the cyclization step in the phosphate-catalyzed reaction are shown in Figure 10. Overall, proton dition,cyclization proton step abstraction in the phosphate-catalyzed by the H2PO4− ion reactionfrom the are(N+1) shown amide in nitrogen Figure 10 occurred. Overall, at proton an early transfer stage transfer and nucleophilic attack for cyclization were similar to those in the syn conformation. In ad- ofand the nucleophilic cyclization step. attack However, for cyclization the H2PO were4− ion similar formed to a those hydrogen in the bond syn conformation.with the amide InNH addition, proton dition, proton abstraction by the H2PO4− ion from the (N+1) amide nitrogen occurred at an early stage ofproton the N-terminal abstraction side. by the The H2 POreaction4− ion mechanism from the (N +of1) the amide anti nitrogenconformation occurred in water- at an early and stagecarbonate- of the of the cyclization step. However, the H2PO4− ion formed a hydrogen bond with the amide NH proton catalyzedcyclization reactions step. However, was also the similar H2PO 4to− thoseion formed of the asyn hydrogen conformation bond with (Figures the amide S6 and NH S7). proton H2O mole- of the of the N-terminal side. The reaction mechanism of the anti conformation in water- and carbonate- culesN-terminal formed side. no hydrogen The reaction bond mechanism with the ofN-terminal the anti conformation main chain atoms in water- in the and water-catalyzed carbonate-catalyzed reac- catalyzed reactions was also similar to those of the syn conformation (Figures S6 and S7). H2O mole- tion.reactions On the was other also similarhand, differences to those of the in synhydrogen conformation bonds between (Figures S6optimized and S7). geometries H2O molecules of syn formed and culesantino hydrogen conformations formed bondno hydrogen with were the not bond N-terminal observed with the mainin theN-terminal chain carbonate-catalyzed atoms main in thechain water-catalyzed atoms reaction. in the Differences water-catalyzed reaction. in On hydrogen- the reac- other tion.bondhand, On formation di fftheerences other might inhand, hydrogen affect differences the bonds activation in between hydrogen energy optimized bondsbarrier. between geometries optimized of syn andgeometries anti conformations of syn and antiwere conformations not observed inwere the not carbonate-catalyzed observed in the carbonate-catalyzed reaction. Differences reaction. in hydrogen-bond Differences formation in hydrogen- might bondaffect formation the activation might energy affect barrier. the activation energy barrier.

Figure 10. Optimized geometries of the cyclization step for the anti conformation in the phosphate-

catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, blue, Figure 10. Optimized geometries of the cyclization step for the anti conformation in the Figurered, and 10. orange, Optimized respectively. geometries Selected of the in cyteratomicclization distancesstep for the are anti in units conformation of Å. in the phosphate- catalyzedphosphate-catalyzed reaction. The reaction. carbon, Thenitrogen, carbon, oxygen, nitrogen, and oxygen, phosphorus and phosphorus atoms are illustrated atoms are in illustrated gray, blue, in gray, blue, red, and orange, respectively. Selected interatomic distances are in units of Å. red,The and optimized orange, respectively.geometries Selectedfor the deammoniationinteratomic distances step are in inthe units phosphate-catalyzed of Å. reaction are shown in Figure 11. As in the cyclization step, the reaction mechanism of the deammoniation step in TheThe optimized optimized geometries geometries for for the the deammoniation deammoniation step step in in the the phosphate-catalyzed phosphate-catalyzed reaction reaction are are the anti conformation was similar to that in the syn conformation. There are no differences in hydro- shownshown in Figure 1111.. As inin thethe cyclizationcyclization step, step, the the reaction reaction mechanism mechanism of of the the deammoniation deammoniation step step in thein gen-bond formation in the deammoniation step between the syn and anti conformations. In addition, theanti anti conformation conformation was was similar similar to that toin that the in syn the conformation. syn conformation. There areThere no diareﬀerences no differences in hydrogen-bond in hydro- distancesformation between in the deammoniation the atoms related step to between reactions the in syn the and anti anti conformation conformations. were In almost addition, the distances same as gen-bondthose of the formation syn conformation. in the deammoniation Therefore, the step syn/anti between conformations the syn and anti do notconformations. affect the mechanism In addition, of distancesbetween thebetween atoms relatedthe atoms to reactions related to in reactions the anti conformation in the anti conformation were almost the we samere almost as those theof same the synas the deammoniation step. In the water- and carbonate-catalyzed reactions, the deammoniation mech- thoseconformation. of the syn Therefore, conformation. the syn Therefore,/anti conformations the syn/anti do conformations not aﬀect the mechanism do not affect of the the deammoniation mechanism of anisms for the anti conformation are considered to be similar to those for the syn conformation (Fig- thestep. deammoniation In the water- andstep. carbonate-catalyzed In the water- and carbon reactions,ate-catalyzed the deammoniation reactions, the mechanisms deammoniation for the mech- anti ures S8 and S9). anismsconformation for the areanti considered conformation to be are similar considered to those to forbe similar the syn to conformation those for the (Figures syn conformation S8 and S9). (Fig- ures S8 and S9).

Figure 11. 11. OptimizedOptimized geometries geometries of the of thedeammoniation deammoniation step stepfor the for anti the conformation anti conformation in the inphos- the

phate-catalyzedphosphate-catalyzed reaction. reaction. The carbon, The carbon, nitrogen, nitrogen, oxyge oxygen,n, and phosphorus and phosphorus atoms atoms are illustrated are illustrated in gray, in Figureblue,gray, blue,red, 11. andOptimized red, orange, and orange, geometriesrespectively. respectively. of Selected the deammoniation Selected interatomic interatomic distancesstep distancesfor the are anti in are units conformation in unitsof Å. of Å. in the phosphate-catalyzed reaction. The carbon, nitrogen, oxygen, and phosphorus atoms are illustrated in gray, The changes in dihedral angles of the main chain in the anti conformation were larger than those blue,The changesred, and orange,in dihedral respectively. angles of Selected the main interatomic chain in distances the anti conformationare in units of Å. were larger than those of the syn conformation (Table5). The amount of alteration for ϕ and ψ from RC to TS1 was 28.1 and of the syn conformation (Table 5). The amount of alteration for φ and ψ from RC to TS1 was 28.1°◦ and 86.8 , respectively. χ was nearly constant throughout the whole reaction. Therefore, large structural 86.8°,◦The respectively. changes in χdihedral was nearly angles constant of the mainthroughout chain in the the whol antie conformation reaction. Therefore, were larger large than structural those ofchanges the syn of conformation the main chain (Table were 5) required. The amount for succinimide of alteration formation for φ and in ψ the from anti RC conformation to TS1 was compared28.1° and changes of the main chain were required for succinimide formation in the anti conformation com- 86.8°, respectively. χ was nearly constant throughout the whole reaction. Therefore, large structural pared with those in the syn conformation. This is considered to be one of the reasons for the low changes of the main chain were required for succinimide formation in the anti conformation compared with those in the syn conformation. This is considered to be one of the reasons for the low

Int. J. Mol. Sci. 2020, 21, 7035 9 of 14 with those in the syn conformation. This is considered to be one of the reasons for the low frequency of deamidation in the anti conformation. In addition, Asn37 and Asn76 of γS-crystallin rarely formed suitable conformations for succinimide formation (Figure S1). Therefore, structural constraints are considered to result in Asn37 and Asn76 rarely being deamidated.

Table 5. Dihedral angles (degrees) of the optimized geometries for the anti conformation in the phosphate-catalyzed reaction.

Dihedral Angle/Degree

ϕ ψ χ ϕH RC 83.20 22.74 138.2 151.8 − − − TS1 111.3 109.5 144.8 171.0 − − − INT1 117.0 133.4 137.1 178.6 − − INT2 114.2 140.1 145.9 179.0 − − − TS2 113.7 142.7 147.2 179.1 − − − PC 113.6 139.6 140.0 178.6 − − −

2.4. Activation Energy for Succinimide Formation from the Asn Syn/Anti Conformation To evaluate the differences in activation energy barrier between the syn and anti conformations, the deamidation pathways of Asn residues were investigated for syn and anti conformations using quantum chemical calculations. The relative energies were calculated using the MP2/6-311+G(2d,2p)// B3LYP/6-31+G(d,p) level (Figure 12 and Figure S10). The energy profiles for two-step succinimide formation from an Asn residue in the phosphate-catalyzed reaction are shown in Figure 12A, B. In both conformations, the relative energy of TS1 was higher than that of TS2. Therefore, the cyclization steps are considered to be the rate-determining steps. The overall activation energies in the syn and anti conformations were 89.3 and 111 kJ mol 1, respectively. Although the activation energy · − barriers in both conformations were significantly low to allow the deamidation reaction to occur under physiological conditions, the barrier of the syn conformation was lower than that of the anti conformation. The activation energy barriers of the syn conformation were in agreement with those experimentally determined for deamidation of model peptides in phosphate buffer at pH 7.5 (90.7 kJ mol 1)[7]. This result suggests that succinimide formation from Asn in the syn · − conformation occurs more easily than in the anti conformation, consistent with experimental data for the deamidation level in γS-crystallin [32]. Furthermore, the activation energies of the syn conformation in carbonate-catalyzed deamidation was 82.7 kJ mol 1 (Figure 12C, D). This value was also consistent · − with activation barriers obtained by experimental methods in various buffers (80–100 kJ mol 1)[6,7]. · − Therefore, H2PO4− and HCO3− ions are considered to be major catalysts for Asn deamidation. Int. J. Mol. Sci. 2020, 21, 7035 10 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 14

AB −1 −1

111 89.3 TS1 TS1 65.7 50.1 TS2 TS2 31.0 20.7 21.5 Relative energy / kJ/ mol energy Relative 15.8 mol kJ / energy Relative INT2 8.73 INT1 PC 0 INT2 2.06 0 RC INT1 PC RC CD −1 −1

91.1 82.7 TS1 TS1 41.7 29.6 28.8 TS2 Relative energy / kJ/ mol energy Relative Relative energy / kJ/ mol energy Relative TS2 17.7 7.44 INT1 0 5.31 0 INT2 INT1 3.21 RC INT2 −13.3 RC PC PC

Figure 12. Relative energy profiles of the phosphate- and carbonate-catalyzed reaction calculated using Figure 12. Relative energy profiles of the phosphate- and carbonate-catalyzed reaction calculated us- the MP2/6-311+G(2d,2p)//B3LYP/6-31+G(d,p) level. Entire energy profiles of (A) syn conformation in ing the MP2/6-311+G(2d,2p)//B3LYP/6-31+G(d,p) level. Entire energy profiles of (A) syn conformation the phosphate-catalyzed reaction, (B) anti conformation in the phosphate-catalyzed reaction, (C) syn in the phosphate-catalyzed reaction, (B) anti conformation in the phosphate-catalyzed reaction, (C) conformation in the carbonate-catalyzed reaction, and (D) anti conformation in the carbonate-catalyzed syn conformation in the carbonate-catalyzed reaction, and (D) anti conformation in the carbonate- reaction are shown. catalyzed reaction are shown. 3. Methods 3. Methods 3.1. Investigation of Asn Conformation in the Experimental Structure 3.1. Investigation of Asn Conformation in the Experimental Structure To investigate the influence of the 3D structure on Asn deamidation rates, an experimental structureTo investigate of γS-crystallin the influence [53] was of the obtained 3D structure from on the Asn PDB deamidation (PDB ID: 2M3T).rates, an To experimental retrieve common struc- turestructural of γS-crystallin features of [53] frequently was obtained deamidated from the Asn PDB residues, (PDB theID: Asn2M3T). conformations To retrieve werecommon observed structural using featuresthe PyMOL of frequently molecular deamidated viewer (http: Asn//pymol.sourceforgen.net residues, the Asn conformations/). were observed using the PyMOL molecular viewer (http://pymol.sourceforgen.net/). 3.2. Molecular Dynamics Simulations 3.2. MolecularTo consider Dynamics structural Simulations flexibility, the MD simulation was performed for the γS-crystallin structure. TheTo system consider was structural solvated with flexibility, the TIP3P the MD model simulation [54], neutralized was performed by adding for Clthe− γion,S-crystallin and calculated structure.under The the system periodic was condition. solvated The with cuto theff TIP3Pdistance model of van [54], der neutralized Waals interactions by adding was Cl set− ion, at 10 and Å,and calcu- the latedparticle under mesh theEwald periodic method condition. [55] The was cutoff used fordistance calculating of van electrostaticder Waals interactions interactions. was The set at SHAKE 10 Å, andalgorithm the particle [56] was mesh applied Ewald to constrainmethod [55] the lengthswas used of bondsfor calculating containing electrostatic hydrogen atoms.interactions. Before The MD SHAKEsimulations, algorithm 1000-step [56] structural was applied minimizations to constrain of waterthe lengths and counter of bonds ion werecontaining performed, hydrogen and 2500-step atoms. Beforeminimizations MD simulations, of whole 1000-step systems were structural performed, minimi followedzations of by water temperature-increasing and counter ion were MD performed, simulation andof 20 2500-step ps with minimizations the temperature of whole raised systems from 0 were to 300 performed, K. After these followed simulations, by temperature-increasing equilibrating MD MDsimulation simulation was of carried 20 ps with out under the temperature constant temperature raised from and 0 to pressure300 K. After for 2000these ns. simulations, The timesteps equili- of bratingtemperature-increasing MD simulation was and carried equilibrating out under MD cons simulationstant temperature were 2 fs. and These pressure MD simulationsfor 2000 ns. wereThe timeaccomplished steps of temperature-increasi using AMBER16 [57ng]. and AMBER equilibratingff14SB force MD fieldsimulations [58] was were used 2 fs. for These the parameters MD simula- of tionsamino were acids. accomplished The cpptraj using module AMBER16 of AmberTools17 [57]. AMBER was ff14SB used force for analysis field [58] of was the results.used for The the rootpa- rametersmean square of amino deviations acids. (RMSDs)The cpptraj for module main-chain of Am atomsberTools17 are calculated was used using for analysis the 3D structures of the results. after The root mean square deviations (RMSDs) for main-chain atoms are calculated using the 3D structures after temperature-increasing MD simulations. The root mean square fluctuations (RMSFs) were

Int. J. Mol. Sci. 2020, 21, 7035 11 of 14 temperature-increasing MD simulations. The root mean square ﬂuctuations (RMSFs) were calculated for all Cα atoms using the MD trajectories for the last 10 ns to evaluate the structural ﬂexibilities. As a reference structure for RMSF calculations, the average structures of the last 10 ns trajectories were used. The dihedral angles ϕ, ψ, χ, and ϕH were investigated using the last 10 ns trajectories.

3.3. Quantum Chemical Calculations Based on the experimental data and the calculated structures of Asn residues in γS-crystallin, the conformations of the initial structure for quantum chemical calculations were constructed. Catalyst molecules and ions used in this study abundantly exist in vivo and have been suggested to catalyze post-translational modification of proteins [41–49]. Energy minima and transitional state (TS) geometries were optimized without any constrains by the density functional theory (DFT) with B3LYP functional and 6-31+G(d,p) basis set in the polarized continuum model (PCM). This method was used in many studies for the reaction mechanisms of post-translational modifications [41–49]. Vibrational frequency calculations were performed for all the optimized geometries to obtain the zero-point energy and to confirm them with no imaginary frequency (for energy minima) or a single imaginary frequency (for TSs). IRC calculations from the TSs to confirm the energy minima connected by each TS. In addition, the single-point energies of all optimized geometries were calculated by MP2/6-311+G(2d,2p) level. All of the quantum chemical calculations were performed using Gaussian16 [59].

4. Conclusions In this study, we computationally investigated the effects of main-chain conformations of Asn residues on the deamidation reaction. MD simulations and quantum chemical calculations demonstrated that Asn deamidation more easily occurs in the syn conformation than in the anti conformation. The activation energy in the phosphate-catalyzed reaction of the syn conformation (89.3 kJ mol 1) was significantly lower than that of the anti conformation (111 kJ mol 1). Therefore, · − · − main-chain conformation is considered to be one of the important factors determining deamidation rate. There is no need for flexibility of the main chain as long as the main-chain conformation is suitable for succinimide formation. Specifically, the dihedral angles ϕ and ϕH are important for determining the deamidation rate of Asn residues. These dihedral angles may be available for prediction of the deamidation rate. On the other hand, common features for χ of frequently or infrequently deamidated Asn residues were not discovered in this study. As side chains exposed to the surface of protein structures are more flexible than the main chains, χ is assumed to be difficult to use as a parameter for predicting Asn deamidation rate. Our results suggest the lifetime of peptide/protein drugs can be extended by excluding syn-conformation Asn residues. In a previous study, the activation barriers of Asp and isoAsp formation from succinimide were shown to be 22.4 kcal mol 1 (93.6 kJ mol 1) and · − · − 22.5 kcal mol 1 (94.1 kJ mol 1), respectively [1]. These values are higher than that of succinimide − · − formation from the syn conformation, and lower than from the anti conformation. Therefore, in the syn conformation, succinimide formation might not be the rate-determining step of Asp/isoAsp formation from Asn residues. Our results also suggest that H2PO4− and HCO3− ions are the principle catalysts for Asn deamidation in vivo, and that an increase in concentration of these anions in cells can accelerate Asn deamidation in proteins. Therefore, computational results in this study suggest the possibility of mitigating age-related diseases by regulating the concentration of H2PO4− and HCO3− ions.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/19/ 7035/s1. Author Contributions: Conceptualization, K.K., E.K. and A.O.; data curation, K.K.; formal analysis, K.K. and T.N.; writing—original draft, K.K.; writing—review and editing, T.N. and A.O. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Grants-in-Aid for Scientiﬁc Research (15H01064), (17K08257) and (19J23595) from the Japan Society for the Promotion of Science. Int. J. Mol. Sci. 2020, 21, 7035 12 of 14

Conﬂicts of Interest: The authors declare that they have no conﬂict of interest to disclose.

References

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