How to engineer ionic liquids resistant enzymes? Insights from

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How to engineer ionic liquids resistant enzymes? Insights from combined molecular dynamics and directed evolution study Subrata Pramanik, Gaurao V. Dhoke, Karl-Erich Jaeger, Ulrich Schwaneberg, and Mehdi D. Davari ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00752 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 31, 2019

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ACS Sustainable Chemistry & Engineering

How to engineer ionic liquids resistant enzymes? Insights from combined molecular dynamics and directed evolution study Subrata Pramanik1, #, Gaurao V. Dhoke1, #, Karl-Erich Jaeger2, Ulrich Schwaneberg1, 3, Mehdi D. Davari1,* 1Institute

of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen,

Germany 2Institute

of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf and

Research Center Jülich, Wilhelm Johnen Strasse, 52426, Jülich, Germany 3DWI

Leibniz-Institute for Interactive Materials, Forckenbeckstrasse 50, 52056 Aachen,

Germany #Shared

first authorship.

*Corresponding author: [email protected]

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Abstract Ionic liquids (ILs) are widely recognized as highly attractive solvents for biocatalysis due to their high stabilities and diverse tunable chemical properties. A major challenge for biocatalysis in ILs is the reduction of enzyme activity. Thus, molecular understanding of enzyme-ILs interactions is crucial to design enzymes for improved ILs resistance. Herein, we studied interactions of Bacillus subtilis lipase A (BSLA) and four commonly used imidazolium-based ILs (1-butyl-3-methylimidazolium (BMIM+) cation with Cl-, Br-, I-, and TfO- anions) using molecular dynamics simulations. Our results show that ILs cosolvents do not alter the overall and local BSLA conformation. However, the ILs effects on the reduction of activity is attributed to dominant surface interactions of BMIM+ cations that strip off essential water molecules from the BSLA surface. Solvent spatial distribution function analysis revealed that BMIM+ has a high binding affinity toward the BSLA surface via hydrophobic or ?1? interactions. Interestingly, the comparison of simulation results with experimental full site saturation mutagenesis BSLA libraries confirmed that most of beneficial positions for resistance improvement are located at the BMIM+ binding regions. These key findings suggest that reducing BMIM+ binding through surface charge engineering might be a general protein engineering strategy to improve BSLA resistance in ILs and is most likely applicable to other lipases and ABC1 *

5

Keywords: Protein engineering, Directed evolution, Biocatalysis, Ionic liquids, Molecular dynamics simulations, Lipase A.

Running title: Bacillus subtilis lipase A in ionic liquids


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Introduction Ionic liquids (ILs) are important solvents for chemo- and biocatalysis 1. ILs are inherited with the ionic nature of inorganic salts and the organic nature of organic solvents that may undergo large structural diversity 2. Their unique properties include good solvation ability, low melting temperature, good conductivity, wide electrochemical window, thermal and electrochemical stabilities, non-volatility, and non-flammability 3-4. Due their properties, IL are highly attractive solvents from a chemo- and biocatalysis point of view

3, 5-7.

ILs are increasingly drawing

attention as green alternatives over volatile organic solvents as reaction solvents 5, 8-9. Recently, ILs based solvent systems have been reported to be promising for the dissolutions of biomass, the fractionation of biomass and the enzymatic depolymerisation of pretreated biomass 10-11. Stability of enzymes in ILs is an essential prerequisite to explore the synthetic application potential of ILs in biocatalysis, for instance performing biomass depolymerisation in homogenous solution

4, 12-14.

Most of enzymes such as lipase

15-17,

cellulase

18,

laccase 7,

xylanase 19, and monooxygenase P450 BM-3 20 tend to deactivate/destabilize in ILs, which may be due to the interaction of ILs with enzymes. Our previous report showed that the residual activity of Bacillus subtilis lipase A (BSLA) was reduced up to 30-40 % in the commonly used BMIM+-based ILs including, [BMIM][Cl] (18.3 % v/v), [BMIM][Br] (13.2 % v/v), [BMIM][I] (10 % v/v), [BMIM][TfO] (15 % v/v), respectively

15.

First principles of enzymes

destabilization in ILs are not well understood and remains challenging for chemo- and biocatalysis in ILs. In general, enzyme activity and stability in ILs rely on several features, including ion-protein contacts, ion-water interactions, inhibitory potential of ions with substrate, protein hydration, and conformation of the active site geometry

19, 21-24.

Previous simulation studies have shown the

interactions of ILs on enzyme stability based on either IL mixtures (e.g. 10-95 wt % of [EMIM][OAC], [EMIM][EtSO4]) 19 , or “pure” ILs (e.g. [BMIM][NO3]) 25. Recently, Kim et al. showed a comparative effect of cations on the activity and stability of Candida antarctica Lipase B (CALB) using [EMIM][TfO], [HMIM][TfO], and [OMIM][TfO]

16.

Kim et al.

attributed the latter findings to the presence of a long hydrophobic tails that facilitate ion-protein interactions and thereby cause structural distortions and a decreased CALB activity in ILs 16. Another study demonstrated the effect of ions by using [C4MIM][Cl], [C2MIM][Cl], [C4MIM][DCA], [C2MIM][DCA] on protein immunoregulatory 7 (IM7) 26. It was reported that size and hydrophobicity of cations are important for the destabilization of IM7, as showed that the interaction of C4MIM+ was more prevalent on denaturation than C2MIM+. Effect of anions showed 3 ACS Paragon Plus Environment


that weakly hydrated DCAN anions strongly bind to positively charged or polar residues, leading to the partial dehydration of the backbone groups, whereas Cl- is the most hydrated anion and should therefore be less available to interact with IM7 26. Likewise, Pfaendtner and co-workers observed that substitution of lysine with glutamate on enzyme surface decreases interactions with the anions; whereas, cation interactions increase with Candida rugosa lipase and Bos taurus A chymotrypsin in [BMIM][Cl] (20 wt%) or [EMIM][EtSO4] (20 wt%) respectively 27. Further studies by Kaar and Nordwald indicated that binding of the BMIM+ cations were decreased (7and 3.5-fold for chymotrypsin from bovine and Candida rugosa lipase, respectively) with increasing ratio of positive to negative surface charges. Same report showed that lowering ratio of positive to negative surface charges leads to preferential exclusion of Cl- anions and increased enzyme stability

28.

Recent NMR studies revealed [BMIM][Cl] induced structural

perturbations near the catalytic triad (S77, D133, and H156) of BSLA, indicating the importance of direct ion interactions with catalytic triad 29. It was showed that substitution of G158E near the catalytic triad resulted in a 2.5-fold resistance improvement of BSLA in [BMIM][Cl] (50% v/v) due to inhibition of the hydrophobic interactions of BMIM+ cations with the catalytic triad. Similarly, another substitution K44E also significantly enhanced resistance of BSLA 29 and was suggested that K44E might diminish the attraction of Cl- anions on the BSLA surface. Additionally, BSLA contains oxyanion hole (I12, M78) that stabilizes the negatively charged reaction intermediates 30. In addition to the catalytic triad, stabilization of oxyanion hole might be essential to improve resistance of BSLA in ILs 31-32. More recent crystallographic 33 and simulation studies 34 described that hydrophobic and

1? interactions of BMIM+ with surface residues

might be a driving force for unfolding and destabilization of lipase in ILs. A recent simulation study from our group demonstrated that hydrogen bond network of the catalytic triad, polarity and shape of the substrate-binding cleft, enzyme hydration and hydrophobic interactions are key features needed to be considered to stabilize BSLA in [BMIM][TfO] 24. The ionic determinants favoring a dominant effect of cation and/or anion remain to be elucidated. While initially the effect of the IL anion on enzyme activity was considered as dominant 35, recent studies demonstrated a more pronounced effect of the IL cation 15, 34. Overall, these observations demonstrate that the influence of ILs on enzymes is complex 24, 33-34, and is not comprehensively understood at the molecular level. In particular, quantitative effect of individual ions, preference of ionic interactions, quantitative effect on hydration level, and interaction energy with enzymes remain to be discovered. Protein engineering is a powerful tool for enzyme stabilization in ILs and first case studies on enzymes with improved IL resistance have been reported and provide first clues to an in-depth understanding of enzymes stabilization in ILs 15, 24, 36. A recent comprehensive studies on the 4 ACS Paragon Plus Environment

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lipase BSLA in which a full site saturation mutagenesis libraries at each positions of the BSLA enzymes was generated and covered all natural amino acid exchanges at each positions concluded that global design principles of enzymes cannot be found in random mutagenesis libraries 15. The comparison of epPCR (random mutagenesis) libraries showed that in directed evolution campaigns only a small fraction of the natural diversity is generated per enzyme position due to the bias of the random mutagenesis methods and organization of the genetic code 37-38. Here we used the data from the site saturation mutagenesis library of BSLA (BSLASSM library) for a comparative analysis and performed molecular dynamics (MD) simulations of BSLA in four ILs cosolvents (ILs cosolvents is referred as ILs throughout the text) to understand how the individual ions affect structure and stability of BSLA at the molecular level. Firstly, we investigated the effect of ILs on structural stability, flexibility and solvent accessibility. Secondly, we described detailed solvation phenomena such as solvents distribution, solvents conformation on BSLA surface, quantitative analysis of solvent interaction on BSLA surface. Next, we compare simulation results with beneficial variants obtained in BSLA-SSM library

15

to validate the computational findings. Finally, we discuss

how the gained knowledge can be used to develop a general protein engineering principle to stabilize ABC1 *

in ILs. Overall, present study provides a molecular insight into the

effect of ILs on BSLA from MD simulations in comparison with BSLA-SSM library to derive a general protein engineering approach to improve resistance of ABC1

*

in ILs.

Methods Molecular dynamics (MD) simulations The starting coordinates of BSLA were taken from the X-ray crystal structure of Bacillus subtilis lipase A (PDB ID: 1I6W chain A, resolution 1.5 Å recently published experimental data

15.

39).

ILs were selected from our

The structures of all four ILs ([BMIM][Cl],

[BMIM][Br], [BMIM][I] and [BMIM][TfO]) are shown in Figure S1 in supporting information (SI). GROMOS96 54a7 force field has been reported to be a reliable force field for simulations of proteins in different cosolvents

40-41

and BMIM+-based ILs

23, 42.

Therefore, we used

GROMOS96 54a7 force field in this study 43. The topologies for BMIM+ cation and TfO- anions were generated using ATB server

44

employing GROMOS96 54a7 force field

43

. Lennard-

Jones (LJ) potential parameters (C6 and C12) for Cl- were taken from GROMOS96 54a7 force field. Whereas, LJ ionic force-field parameters for Br- and I- were adapted from the OPLS-AA force field

45-46.

The R and S values were converted to C6 and C12 for consistency with 5 ACS Paragon Plus Environment


GROMOS96 54a7 force field

47-49,

as previously reported

50-51.

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In order to validate ILs force

field parameters, triplicate 10 ns MD simulations were performed using pure ILs without BSLA to determine reproducibility of experimental density (details are provided in the “Validation of ILs force field parameters” section in SI). It was observed that ILs densities obtained from MD simulations are highly correlated with experimental densities of all pure ILs (Table S1 in SI). Moreover, we observed that density of [BMIM][TfO] obtained from simulations using GROMOS96 54a7 force field is highly similar with density obtained from simulations using Amber ff99SB force field reported by Kim et al.

16

(Table S1 in SI). We further analyzed

representative coordinates of all ILs after 10 ns MD simulations. It was observed that ILs ions do not form aggregate for all ILs and ILs ions remained homogeneously distributed thought the simulation box (Figure S2 in SI). The protonation states were assigned based on calculated pKa using PROPKA method

52

and employing the PDB2PQR server

53.

catalytic triad was considered based on catalytic mechanism of lipase

Protonation state of

36, 54,

specifically, the

protonation state was assigned to LV atom of catalytic residue H156 based the proton transfer mechanism involved in an activation of the hydroxyl group of the catalytic serine of the catalytic triad (S77, D133, and H156) 36, 54-55. Hydrogens were added to the protein molecule by using pdb2gmx application in GROMACS 5.1.2. The protein molecules were placed in a cubic simulation box (10 nm3). The ILs were prepared according to experimental conditions ; i.e. 18.3 % v/v [BMIM][Cl], 13.2 % v/v [BMIM][Br], 10.0 % v/v [BMIM][I], 15.0 % v/v [BMIM][TfO] in water. BSLA retains 35.0, 39.0, 37.0, and 30.0% of it residual activity in these concentrations, respectively 15. Single Point Charge (SPC) water model was used 49, 56. In order to neutralize the system, the Cl-, Br-, I- and TfO- were added into simulation box for [BMIM][Cl], [BMIM][Br], [BMIM][I] and [BMIM][TfO] cosolvents, respectively. For simulation of system in water only, Cl- anions were added to neutralize the system. The electrostatic interactions were calculated by applying the particle mesh Ewald (PME) method 57-58.

Short-range electrostatic interactions and van der Waals (vdW) were calculated using a

cut off value 1.0, respectively. Energy minimization of the whole system for each ILs was performed individually using steepest descent minimization algorithm until the maximum force reached to 1000.0 kJ mol-1 nm-1. Subsequently, system equilibration was performed under NVT and NPT ensemble. First, NVT equilibration was conducted at constant temperature of 300 K for 100 ps with time step of 2 fs. Initial random velocities were assigned to the atoms of the molecules according to the Maxwell–Boltzmann algorithm at same temperature. Second, NPT equilibration was conducted at constant temperature of 300 K for 100 ps with time step of 2 fs, respectively. The Berendsen thermostat

59

and Parrinello-Rahman pressure 6

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60

coupling were

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used to keep the system at 300 K, time constant :XT) of 0.1 ps and 1 bar pressure, time constant :XP) of 2 ps. The production run was carried out in triplicate using NPT ensemble for 100 ns with time step of 2 fs at constant temperature of 300 K. All bonds between hydrogen and heavy atoms were constrained with the LINCS algorithm. All the calculations were carried out using GROMACS 5.1.2

61-62

(details are provided in the “analysis of MD simulation trajectories”

section in SI). Visualization and analysis were performed using VMD 1.9.1 63 and GROMACS tools

61-62.

The binding free energies between molecules were calculated using MM/PBSA

method using g_mmpbsa tool 64 in GROMACS as applied previously 65-68.

Analysis of BSLA-SSM library To compare MD simulations results with BSLA-SSM library

15,

we analyzed the effect of

substitutions on resistance of BSLA in four ILs. The substitutions were analyzed to identify the general patterns for beneficial substitutions (location, type of amino acid exchange) in the 3D structure of BSLA. The detailed analysis including (i) overview of beneficial substitutions, (ii) number of beneficial amino acid substitutions, and (iii) location of beneficial amino acid substitutions in the BSLA 3D structure are provided in “Analysis of BSLA-SSM library” section in SI.

Results and Discussion The main objective of this study is to understand the interaction of BSLA and ILs at molecular level to provide a general protein engineering approach to improve resistance of enzymes in ILs. The results section is organized as follow: first, we analyzed overall structural stability, flexibility and solvent accessibility of BSLA in ILs. Then, we describe detailed solvation phenomena including solvents distribution on the BSLA surface, solvents conformation on the BSLA surface, quantitative analysis of solvent interaction in the hydration shells. Finally, to identify the main driving force for BSLA and ILs interactions, we calculated non-bonded interactions (electrostatic and vdW) energy of water and ILs ions with BSLA.

BSLA structure remains stable in ILs The structural stability of BSLA in ILs was evaluated based on the analysis of backbone root mean square deviation (RMSD) of overall structure. All simulations converged after ~60 ns yielding final RMSD values of ~1.15-2.00 Å, as observed from three independent 100 ns 7 ACS Paragon Plus Environment


simulations (Figures S3, S4 in SI)

69-70.

Clustering analysis showed that most populated

cluster of RMSD was observed at 1.16 Å in water, whereas it was slightly varied within 0.921.53 Å for ILs (Figure S5 in SI). These observations indicate that the BSLA structure remains stable in binary mixtures of ILs and water at reported experimental concentrations (i.e. 18.3 % v/v [BMIM][Cl], 13.2 % v/v [BMIM][Br], 10.0 % v/v [BMIM][I], 15.0 % v/v [BMIM][TfO] in water)

15.

Furthermore, to gain a better understanding of the localized

dynamics of the protein, we determined the average root mean square fluctuation (RMSF) per residue from 60-100 ns (after convergence) for protein. RMSF analysis showed slightly higher flexibility of BSLA (Figure 1A) structure in the presence of ILs compared with water especially in the loop-helix transition regions (residues 45-51, 106-116, 135-141, Figure 1). The A1

@ and C-sheet showed less flexibility in all solvents. Higher structural changes

were observed in the [BMIM][I] compared with other solvents. It is noteworthy that the flexibility enhanced regions (e.g. M78, M134, Y161) are located in close proximity to the catalytic triad (S77, D133, and H156) (Figure 1). Radius of gyration (Rg) analysis showed a partial swollenness in ILs compared to water (Figure S6 in SI). We observed number of intra-protein H-bonds partially reduced and salt-bridges network were also partially impaired in the presence of ILs (except [BMIM][TfO]) (Figures S7-S8 in SI and Tables S2-S6 in SI).

H156

A RMSF (Å)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4

— Water — [BMIM][Cl] — [BMIM][Br] — [BMIM][I] — [BMIM][TfO]

D133

B S77

135-141

3

45-51

2 1 0 1

21

41

61

81

101

121

141

161

181

106-120 Residue

Figure 1. Residue wise flexibility of BSLA based on RMSF (Å). (A) Average RMSF from three independent simulations (last 40 ns trajectories) of BSLA in water and ILs showed that flexibility is slightly increased in certain loop region (labeled in magenta). (B) Locations of the most flexible regions are shown in magenta color. Catalytic residues, S77, D133, and H156 are less flexible and shown in green color.

As mentioned earlier, the computational analysis suggests that the BSLA structure remains stable in ILs, therefore, we further determined the solvent accessibility surface areas (SASAs) 8 ACS Paragon Plus Environment

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of BSLA to investigate contact area of BSLA with solvents. It was found that total the solvent accessible surface area was increased in presence of ILs compared to water (Figure 2). Likewise, hydrophobic and hydrophilic SASAs also showed a similar trend in which hydrophobic interactions was increased in ILs than water.

9500

Total

Hydrophilic

Hydrophobic

9000

SASA (Å2 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8500 5500 5000 3700 3200

Figure 2. Average of total, hydrophobic, and hydrophilic SASAs

71

of BSLA from three

independent simulations trajectories (from last 40 ns) in water and ILs are shown. All SASAs are significantly increased in ILs when compared water. Specifically, hydrophobic contact areas were highly increased in presence of ILs.

BMIM+ ions dominate the interactions with the BLSA surface In order to understand the influence of ILs on the first hydration shell, spatial distributing functions (SDF) of water and ILs ions were calculated. We determined region and density of water and ILs on the BSLA surface based on SDF analysis as shown in Figure 3. Comparison of SDF in water and ILs show that the water is distributed in the similar regions over the BSLA surface in all ILs except [BMIM][TfO]. A closer look at Figure 3A in comparison with Figure 3B-E indicates that water distribution was reduced in the presence of ILs which signifies that the hydration shell of the enzyme was distorted in the ILs. Figure 3B-D clearly showed that interaction of BMIM+ was predominant compared to anions (Cl-, Br- and I-) in case of halogenated-[BMIM]. Density of Cl- anions was lower than Br- and I- on the BSLA surface and ClN does not show significant interactions with the BSLA. The I- anions interact more compared with Br-. Distribution of I- was near to the catalytic triad in which positively charged H156 is located. These results 9 ACS Paragon Plus Environment


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revealed that BMIM+ ions interact more favorably with the BSLA surface in case of halogenated-BMIM, which might play a dominant role in reduction of the catalytic activity. This observation suggests that reduction of BMIM+-binding to the BSLA surface might be a promising approach to stabilize BSLA in ILs (details analysis is shown in “Analysis of BSLASSM library” section in SI and as will be discussed further in “Discussion” section) 15. The effect of TfO- anions remain critical because it showed a much higher tendency to interacts with the BMIM+ cations on enzyme surface than Cl-, Br-, and I-. As it can be clearly seen in Figure 3E, TfO- anions distribution was almost equal with BMIM+ cations on BSLA surface. The effect of anions may vary depending on their size and hydration level which might lead to higher electrostatic and counter ions effects of TfO- anions with BMIM+ cations than halogen anions 26. Due to higher electrostatic and counter ions effects, the TfO- anions and BMIM+ cations interacts with similar regions of the BSLA surface and exhibits similar patterns. Likewise, solvent distribution shows that the density of BMIM+ is much higher near to the catalytic triad and oxyanion hole regions indicating an affinity of the BMIM+ molecules towards these regions (Figure 3). Since, the oxyanion hole stabilizes the catalytic triad during the formation of reaction acyl-enzyme tetrahedral intermediates 30, binding of BMIM+ may also lead to the reduction of enzymatic activity.

A

Water

B [BMIM][Cl]

C [BMIM][Br]

D [BMIM][I]

E [BMIM][TfO]

180°

Water

Cation

Anion

Catalytic triad

Oxyanion hole

Figure 3. SDFs for the solvent (water, ILs ions) distribution of the BSLA surface in water and ILs simulations. (A) Distribution of water is shown for simulation in water (Isovalue 15.5). It can be observed that some water molecules bind to the oxyanion hole. (B) Distribution of water (Isovalue 17), Cl- (Isovalue 75) and BMIM+ (Isovalue 19) is shown for simulations in [BMIM][Cl]. (C) 10 ACS Paragon Plus Environment

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Distribution of water (Isovalue 15.5), Br- (Isovalue 250) and BMIM+ (Isovalue 33) is shown for simulations in [BMIM][Br]. (D) Distribution of water (Isovalue 15.5), I- (Isovalue 160) and BMIM+ (Isovalue 29) is shown for simulations in [BMIM][I]. (E) Distribution of water (Isovalue 15.5), TfO(Isovalue 100) and BMIM+ (Isovalue 65) is shown for simulations in [BMIM][TfO]. Color code used: grey for enzyme surface, orange for oxyanion hole, green for catalytic triad, blue for water, purple for BMIM+, and cyan for anions.

ILs ions strip off the essential water molecules from BSLA surface Analysis of SDF provide distribution of water and ions on the BSLA surface. Based on this observation, we further quantified the number of water and ion molecules within first solvation shell around the BSLA (BMIM+ and TfO- showed first solvation shell at ~6.5 Å whereas first solvation shell was observed within ~2.25 Å for halogen anions). Figure 4 shows a noticeable difference between the number of water molecules in the first hydration shell around the BSLA surface in only water and ILs (Figure 4A), with a significant reduction of water molecules in ILs when compared to only water simulations. This observation indicates that retention of surface water molecules might be essential to stabilize BSLA in ILs 72. Therefore, it seems that reduction of surface water molecules in the presence of ILs might lead to the reduction of BSLA activity. Consequently, we further quantified the number of cations and anions interactions with the BSLA surface (Figure 4B). 30-40 BMIM+ cations were in case of halogenated-BMIM interacting within the first solvation shell the BSLA surface. Number of halogen ions remained significantly low, whereas the number of TfO- on the surface showed almost an equal number of interactions as [BMIM]+. The binding intensity of anions was TfO->I->Br->Cl-. These observations are consistent with the findings from SDF analysis, in which we observed reduction of surface water and major interactions of BMIM+ with BSLA. Additionally, we quantified the number of water molecules and ions interacting with the catalytic triad (Figure S9A in SI). It was observed that the number of water molecules were not changed in ILs in comparison with water only simulations. Whereas, BMIM+ ions predominantly interact with the catalytic triad in case of halogenated-BMIM, in which both ions interact with catalytic triad for [BMIM][TfO] (Figures 3E, S8B in SI).



A

B

450

60

Cation Number of cations or anions

400

Number of water molecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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350 300 250 200 150 100

Anion

50 40 30 20 10

50 0

0 Water

[BMIM][Cl] [BMIM][Br]

[BMIM][I] [BMIM][TfO]

[BMIM][Cl]

[BMIM][Br]

[BMIM][I]

[BMIM][TfO]

Figure 4. Average number of water molecules and ILs ions interacting within first solvation shell of the BSLA surface. (A) Average numbers of surface water molecules within the first solvation shell of the BSLA surface were significantly reduced in ILs in comparison with only water. (B) BMIM+ cations (30-40 molecules) mainly interact with the BSLA surface in case of halogenated-BMIM and number of halogen ions were significantly less on the BSLA surface. Equal number of cations and anions interact with the BSLA surface in case of [BMIM][TfO].

Hydrophobic and H'H interactions drives the BMIM+ binding to BSLA surface To determine surface contact of ILs with BSLA, orientation conformations of ILs ions with BSLA surface were calculated by analyzing radial distribution functions (RDFs) as shown in Figure S10 in SI. The position of the peaks in the cation-BSLA surface are almost identical and first solvation shell was observed through hydrophobic interaction of BMIM+ for all ILs. The first solvation effect from RDF showed that hydrophobic tail (C1 atom of BMIM+) interacts with BSLA and the order of interactions was observed as C1>C3>C7 (Figure 5A and Figure S10 in SI). The positions of the peaks are identical and appear within ~5 Å in all ILs and first solvation shells were limited with ~6.5 Å. Comparatively a minor difference in the solvation effects of BMIM+ varied based on the associated anions which can be observed from the heights of the peaks (Figure S10 in SI). Solvation of TfO- showed that first solvation was due to binding of C1 atom with BSLA. The height of peak reflect that interaction of TfO- is quite similar as with BMIM+. For halogen ions, first solvation shell was observed within ~2.25 Å (Figure S11 in SI). These observations showed that BMIM+ interacts with BSLA through hydrophobic interactions. Additionally, aromatic residues including Y139, W42 showed ?-? interactions of BMIM+ (Figure 5C-D and details in Figures S12S15 in SI). As water interactions with enzyme play a critical role in determining enzyme structure, function, folding properties, orientation of oxygen and hydrogens atoms (Owater and Hwater) of water 12 ACS Paragon Plus Environment

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with BSLA surface based on RDF (Figure S16 in SI) showed that both Owater and Hwater have almost equal preferential of interaction on the BSLA surface. First hydration shell was observed within ~1.8 Å from the BSLA surface.

A

C7

1.5

H 3C N

+

B 1.5

N1 C3

C1

N

CH3

C H2

1

1

g(r)

g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5

0.5

0

0 2

C

-

4

6 r(Å)

S1 O C1 F

O S C F O F 2

8

4

6

8

r(Å)

D

Y139

W42

Figure 5. (A) RDFs of BMIM+ in [BMIM][Cl] showed that C1 atom interacts with the BSLA and the order of interactions was observed as C1>C3>C7. Similar RDFs for BMIM+ was observed for other ILs as shown in Figure S10 in SI. (B) RDFs of TfO- in [BMIM][TfO] showed that C1 atom interacts with BSLA followed by S1 atom. (C-D) Aromatic residues Y139 and W42 showed ?-? interactions (cut off 5 Å) with BMIM+ in [BMIM][Cl]. These residues also showed ?-? interactions in other ILs. Details ?-? interactions are shown in Figures S12-S15 in SI.

Additionally, we calculated binding energy of each solvent molecule to the BSLA and its catalytic triad. It can be observed that water molecule shows stronger interaction with BSLA in all solvents (Table S7 in SI). For anions, electrostatic interaction was the main driving force of interaction. Besides, vdW also has major contribution in case of TfO- (-185.94 ± 40.22 kcal/mol), whereas it was unfavorable in case of Cl-, Br-, and I- anions. Interaction of BMIM+ showed an opposite behavior compared with anions. The vdW was the main driving force of BMIM+ interaction for all ILs and electrostatic remains less favorable to interacts with the BSLA surface (Table S7 in SI). This observation reflects that hydrophobic interactions might 13 ACS Paragon Plus Environment


be a key factor for BMIM+ interactions with aliphatic residues on the BSLA surface 15. As we observed from SDFs that ions interact towards catalytic triad (Figures 3, 4), we further calculated electrostatic and vdW non-bonded interaction energies between ILs and catalytic triad (i.e. S77, D133 and H156). Initially, effects of each ion were determined with total catalytic triad to reveal overall interactions with catalytic residues (Table S8 in SI). Secondly, ionic effects with each residue, including S77, D133 and H156 were determined to understand their contribution on non-bonded interaction energy (Tables S9-S11 in SI). In case of catalytic triad, higher binding energy was observed for BMIM+ cations, whereas anions show weaker interactions with enzyme except TfO- (vdW = -3.33 ± 1.31) (Table S8 in SI). In case of halogenated-[BMIM], electrostatic and vdW energies changes over time and showed similar pattern for both cation and anions. Nevertheless, BMIM+ showed relatively weak interactions in case of [BMIM][Br] and TfO- interacts much stronger than halogen anions. As shown in Table S8 in SI, electrostatic energy between catalytic triad and BMIM+ in all solvents is much higher than vdW. Thus, BMIM+ cations play an important role in enzyme activity due to strong electrostatic interactions with the catalytic triad of BSLA, which may explain that effect of anions was overcompensate by the effect of cations (Table S8 in SI) 26. Contribution of each catalytic residue, S77, D133 and H156 showed that D133 plays a pivotal role in the binding with all ILs (Tables S9-S11 in SI). S77 has unfavorable electrostatic energy with BMIM+ (except [BMIM][Cl]), whereas it showed weak vdW interactions with BMIM+. Oppositely, S77 showed favorable electrostatic interactions with anions, except Cl- anion (Table S9 in SI). In case of D133, it was observed that binding of BMIM+ cations with D133 was the main driving force of interaction (Table S10 in SI). D133 mainly interacts with BMIM+ cations through electrostatic interaction and vdW remains less favorable. D133 interaction with anions remain unfavorable as shown in Table S10 in SI. In case of H156, both anionic and cationic effect remain interesting, in which both ions have quite strong interaction energies for [BMIM]TfO], but not with other ILs (Table S11 in SI). Regarding binding energy results analysis, it is important to discuss the influence of ILs dielectric constant on the possible error for estimation of non-bonded interactions. According to MM/PBSA method, default solvent dielectric constant was taken as 80 (water) 73, whereas dielectric constants of pure ILs are 11-20 74-75. In this work, default solvent dielectric constant was used in the calculation for mixture of ILs and water. Therefore, a relatively higher dielectric constant of ILs might lead to the strong electronic polarization of the binding interface (Tables S7-S11 in SI) 76-77. This observation is consistent with the previous studies in which the higher electrostatic effect was observed due to higher dielectric constant 74-78. 14 ACS Paragon Plus Environment

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Having presented the MD simulation results, we discuss in details the implication to improve resistance of lipases and ABC1 *

through directed evolution in the following sections.

Excellent substrate solubilization properties of ILs are important for chemo- and biocatalysis. Enzymes such as lipases have various important role in different industrial activities through the application of bioprocess technology 3, 8, 79-80. A major drawback of using ILs in biocatalysis is enzymes often lose activity at high ILs concentration (>10 %) 15, 80. Stabilization of enzymes in ILs through protein engineering as well as development of novel ILs depend on the fundamental understanding of the specific interactions of ILs ions with enzyme at molecular level. Our previous study showed ILs reduce BSLA activity in ILs, further site saturation mutagenesis on the each position of BSLA discover that (i)

1.F_G of all positions and (ii) 6-

_G of substitutions contributed to improve resistance of BSLA in ILs

15.

However, the

molecular effect of ILs and mechanisms for improved resistance of beneficial variants at molecular level is not well understood. We deciphered in details the molecular effect of ions, water on BSLA using MD simulations.

BSLA structure remains stable and essential water stripped out from the BSLA surface By analyzing several structural features, including protein overall stability, compactness, and residue-specific flexibility, present study demonstrates that overall conformation of BSLA remained stable in BMIM+-based ILs having concentrations ~10-19 % v/v 15. In general, global structure remained stable in these ILs (at concentrations: ~10-19 % v/v) from three 100 ns simulations and there are not a significant correlation between structural stability and flexibility with the experimentally observed activity of BSLA

15, 29, 34, 81.

As structure and function of

biomolecules can be strongly influenced by their hydration shells, we analyzed the solvation mechanism of BSLA in water only and the different ILs simulations 72, 81. Effects of ILs on the reduction of activity is attributed to dominant surface interactions of BMIM+ cation and stripping off essential water molecules from the BSLA surface. Analysis of SASAs showed that BSLA solvent accessibility was enhanced in the presence of ILs compared to water only simulations. This effect may be attributed to higher interactions of ILs with the BSLA surface than water 82. The cations moiety in these BMIM+-based ILs play a very important role in the interactions of ILs with BSLA through hydrophobic and ?-? interactions. Interactions of ILs ions varied based on combinations of cation and anion. Our study shows that the main reason for reduction in the BSLA activity is (i) surface interaction of BMIM+ cation and (ii) disrupting the hydration shell by stripping off essential water molecules. This observation indicates that 15 ACS Paragon Plus Environment


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BMIM+ cation majorly penetrates into the water layer and interacts with BSLA by hydrophobic, and ?-? interactions and thereby altering dynamics of water molecules in the hydration shell which resulted in the reduction of activity 72. Moreover, BMIM+ might further interacts directly or indirectly with substrates, thus might lead to a competitive inhibition of substrate accessibility and thus reduction of activity. With respect to Hofmeister effects, the order of reduction of activity is TfO->I->Br->Cl- which is in consistence with our previous experimental study 15. Higher effect of TfO- might be possible due to two major reasons: (i) delocalize charge distribution of TfON and weaker solvation shell, which can therefore easily lead to form Hbonds with amides of the enzyme backbone than halides anions and (ii) a single TfON might form multiple H-bonds with amino acids of BSLA and disturb the enzyme structure more effectively than halides anions 15, 83-84. We observed strong electrostatic interaction than vdW (Table S7 in SI) interaction between catalytic triad and BMIM+ cations and the anions interactions remained unfavorable (Tables S8-S11 in SI). It is reported that non-bonded interactions between enzyme and ILs can affect the conformational changes and consequently enzyme stability 16-17, 77, which is determined by an electrostatic and vdW interactions. These observations are in consistence with effect of ions particularly observed from solvation mechanism where organic BMIM+ cations showed strong affinity toward enzyme surface, specifically near the catalytic triad and oxyanion hole. Our results are also in agreement with the earlier reports

18, 26, 29, 33-34, 85-86,

demonstrating that the

organic cations of ILs favor protein destabilization unless the cation effect is overcompensated by the anion effect. However, effect of cation/anion and their destabilization mechanism depends on the different combinations of cations and anions 16-17, 26, 85. Therefore, depending on combination of ions and by understanding their effect, surface charge modification of enzyme might be a rational approach to stabilize enzyme in IL environments 85, 87-89.

Improved resistance variants from BSLA-SSM library corroborate with the reduction of BMIM+ binding Based on the solvation mechanism, it has been observed that BMIM+ majorly interacts with the BSLA surface through hydrophobic and ?-? interactions (Figure 6) and plays a dominant role in the reduction of BSLA activity 24, 29. These observations lead to a hypothesis that reduction of BMIM+-binding to BSLA surface might be an attractive approach to stabilize lipase in ILs. To confirm this hypothesis, we performed a comparative analysis of previously identified beneficial substitutions from the BSLA-SSM library 15. This library showed that 50–.F_G of 16 ACS Paragon Plus Environment

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the amino acid positions contributed to improved resistance of BSLA in ILs. The wild type BSLA contained 127 surface exposed residues (water probe radius 1.5 Å, cutoff of ` _G; 15. Further analysis revealed that ~70-90% beneficial substitutions are surface exposed (Table S12 in SI). Specific analysis showed that 36-63 % (out of 127 positions) surface-exposed positions substituted with polar and/or charge residues improved resistance of BSLA in ILs as shown in Table S13 in SI. Surface exposed aliphatic amino acids are most predominant positions (3844%), lead to resistance improvement of BSLA in ILs through polar and/or charge substitutions. In the wild type BSLA, amino acid composition consists of " 5._G aliphatic, F5E_G polar, E5 _G charged, and E5 _G aromatic which is preferable for its catalytic performance in aqueous solution. Whereas, substitutions contributed to the resistance of BSLA in ILs showed that improved variants were obtainable by substitution to polar : E1 "_G; charged :

1

_G;

aliphatic : E1 _G; and aromatic : !1 F_G; amino acids. These observations demonstrate that substitutions of aliphatic amino acids with polar and/or charge amino acids on the surface is a general trend to improve resistance of BSLA in ILs

15.

Our simulations showed that

hydrophobic interactions are major driving force of BMIM+ interactions (Figures 3, 4), indicating that surface exposed aliphatic amino acids are highly prone to interacts with BMIM+. In the Figure 6, a closer look on beneficial positions and BMIM+ binding sites (from SDF analysis) clearly shows that locations of beneficial positions are overlapping well with BMIM+binding regions (Figure 6 and Figure S17 in SI for details) 15. We observed that a significant number (~50 %) of beneficial substitutions 15 are located towards the BMIM+ binding sites for all ILs. This observation verifies that substitutions toward BMIM+ binding region might interfere and reduce hydrophobic BMIM+ interactions on the surface, which subsequently disrupt local hydrophobic networks 15, 29. However, it is also crucial to note that enzyme stability in ILs is affected by multiple factors such as H-bonds, salt-bridges, surface water network, hydration shell, hydrophobic interactions 24, 27, 85.



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B

A

180°

Figure 6. Comparative analysis is shown for (A) BMIM+ binding sites and (B) beneficial positions substituted with polar and/or charge residues from BSLA-SSM library

15

for

[BMIM][Cl]. It was observed that 80 positions out 127 surface-exposed positions are beneficial and contributed to BSLA resistance in [BMIM][Cl]. This comparison showed that BMIM+ binding sites are overlapped with beneficial positions, indicating that substitutions at these positions might lead to reduction of BMIM+ binding and thus improved resistance of BSLA in ILs. A similar overlapping of BMIM+ binding sites and beneficial positions was observed in other ILs as shown in Figures S17, S18 in SI. Used color codes are grey: enzyme surface, orange: oxyanion hole, green: catalytic triad, purple: BMIM, and cyan: beneficial positions.

A general strategy to stabilize IJK'

in ILs

In conclusion, the obtained results revealed that overall and local conformation of BSLA remains stable in ILs; stripping off essential water molecules from the BSLA surface and interaction of BMIM+ cations with surface are the key factors contributing to the reduction of BSLA activity in ILs. Based on these findings and comparison to experimental data, we propose that reduction of BMIM+ binding affinity through surface charge engineering towards the BSLA surface might be a general strategy to stabilize BSLA and other enzymes sharing a similar ABC1 hydrolase in ILs.


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Acknowledgment Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects JARA0187.

Conflict of Interest The authors declare that they have no conflict of interest.

Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: Methods for validation of ILs force field parameters, analysis of MD simulations trajectories, analysis of BSLA-SSM library; table of ILs densities, tables of salt-bridges; tables of nonbonded energies; tables of amino acids substitutions and their positions; figure of chemical structures of ILs; figures for structural stability and flexibility of BSLA; figures for ?–? interactions; figures for comparative analysis of BMIM+ binding regions and beneficial positions.

Author information Corresponding Author: [email protected] (M.D.D.). ORCID Subrata Pramanik: 0000-0003-3328-6239 Gaurao V. Dhoke: 0000-0002-2294-5799 Karl-Erich Jaeger: 0000-0002-6036-0708 Ulrich Schwaneberg: 0000-0003-4026-701X Mehdi D. Davari: 0000-0003-0089-7156



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