pH-Induced Rotation of Lidless Lipase LipA from Bacillus subtilis at

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pH-Induced Rotation of Lidless Bacterial Hydrophobin Bacillus subtilis lipase A at Lipase-Detergent Interface Sudip Das, and Sundaram Balasubramanian J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02296 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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The Journal of Physical Chemistry

pH-induced Rotation of Lidless Bacterial Hydrophobin Bacillus subtilis lipase A at Lipase-detergent Interface Sudip Das and Sundaram Balasubramanian∗ Chemistry and Physics of Materials Unit Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India E-mail: [email protected]

∗

To whom correspondence should be addressed

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Abstract Lipases exhibit a unique process during their catalysis of the hydrolysis of triglyceride substrates, called interfacial activation. Surfactants are used as co-solvent with water not only to offer a less polar environment to the lipases needed for their interfacial activation, but also to solvate the substrate which are poorly soluble in water. But, the presence of detergent in the medium can affect both the lipase and the substrate, making the construction of a microkinetic model for lipase activity in the presence of detergent difficult. Herein, we study the interfacial activation of a lidless lipase Bacillus subtilis lipase A (BslA) using extensive atomistic molecular dynamics simulations at different concentrations of the surfactant, Thesit (C12E8) at two pH values. Residues which bind to the monomeric detergent are found to be the same as ones which have been reported earlier to bind to the substrate. Very importantly, a pH-induced rotation of the enzyme with respect to surfactant aggregate has been observed which not only explains the experimentally observed pH-dependent enzymatic activity of this lidless lipase, but also makes BslA a switchable Janus nanocolloid with potential applications in drug delivery, electronics and food industries.

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Introduction Lipases catalyze the hydrolytic cleavage of ester bonds of triglycerides. The residues constituting the active site in esterase and lipases are similar, so also are the substrates. But lipases have drawn considerable attention of researchers, 1–7 mainly due to their unique behavior called interfacial activation which is not observed in esterases. When a lipase approaches the aqueous-lipidophilic interface (formed by substrate and/or surfactant aggregates), it opens up its flexible region (often called as ’lid’) and gets activated. 8–12 This process is termed as interfacial activation. In most cases, lipases prefer a less polar and a more hydrophobic environment and thus the stability and activity of lipases in many non-aqueous solvents (e.g. organic solvent, ionic liquid or surfactant in water) have been studied. 8,11,13–39,39–49 Furthermore, detergents are used to solvate the substrate (mainly lipids) which are either sparingly soluble or insoluble in water. 37–39 The presence of detergent in the medium can affect both the lipase and the substrate in multiple ways, making the construction of a microkinetic model for lipase activity in the presence of detergent almost impossible. 8,11,39–49 The mechanism of interfacial activation of lipases possessing a lid domain has been explored well; the conformational change of this lid is found to be the most crucial step behind their activation. 8–12,36,50 However, the interfacial activation of lidless lipases has yet been largely unexplored. In this situation, it is very important to establish a microkinetic model for interfacial activation and the catalytic mechanism of lipases without any lid and in the presence of surfactant. For such studies, Bacillus subtilis lipase A (BslA) can be a perfect model, as it is the smallest lipase (containing only 181 amino acid residues) and one which does not contain a lid. 51–54 It is a minimal α/β-hydrolase fold enzyme consisting of six β-strands in a parallel β-sheet, surrounded by five α-helices. 51 There are three α-helices on one side of the β-sheet and two on the other side (Figure 1 (a) and (b)). Like other lipases, the active site of BslA is also formed by a catalytic triad (S77, H156 and D133) and an oxyanion hole (I12 and M78). 3



Figure 1: (a) Secondary structure of BslA (white cartoon) with its active site residues (represented as licorice), head region (yellow cartoon) and α1-helix, β2-sheet and α2-helix (brown cartoon). (b) Another view of (a). BslA had been found to coexist in two different conformations in its crystal structure (PDB ID: 4bhu 55 ). In one of them, the side chains of residues near the active site (L76, L77, L79, G80, L119, L121, L123, L124, L153 and I155) are oriented outwards, while in the other form, the side chains of these residues are buried inside the protein. To describe the orientation of BslA near an oil-water interface, Brandani et al. 56 termed these conformations as Lout and Lin , respectively. They found that BslA exists in the Lout conformation where the residues near the active site, termed as ’hydrophobic cap’, were pointed towards the oil phase. But by varying the conditions (say, temperature, pressure or the lipase’s local chemical environment) which enables the Lout to Lin conformational transition, the whole enzyme was seen to rotate so as to facilitate the movement of the hydrophobic cap from being oil-faced to a water-faced orientation. These two aspects, i.e., the enzyme rotation and the absence of any lid make BslA an interesting candidate to investigate the mechanism of interfacial activation for lidless lipases in the presence of surfactant. Another important aspect of BslA has recently been exposed by Hobley and co-workers. 55,57 They found BslA to self-aggregate to form hydrophobic biofilms around Bacillus subtilis bacteria to protect the bacterial community from the external aqueous medium, making

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BslA a bacterial hydrophobin. 55,57 Fungal hydrophobin, a Janus nanocolloid, has been widely used for decades in electronics, medicine and food industries. 58–65 The behavior of BslA too as a hydrophobin could enable its use in such industries. Like fungal hydrophobin, 66–70 one of the significant applications of BslA could be as a biofilm coating formed by its self-aggregation over drug carrier nanomaterials to increase the biocompatibility and bioavailability of lipophilic drugs. For the efficient release of drugs at the targeted cells, the BslA coating should be broken at appropriate conditions. Detergents can be a good choice to disintegrate this coating; especially polyethylene glycol monododecyl ether (C12E8), which has been employed for a long time as an efficient membrane solubilizer. 71–73 It should be noted that polyethylene glycol, the head group of Thesit (C12E8), has been found to impart in vivo longevity to drug carriers. 74 The importance of studying the BslA-C12E8 combination can lead to a molecular level understanding of the interfacial activation of lidless lipases. A pioneering work in this regard is that of Kubler et al. 75 They chose BslA with a non-polar detergent Thesit and studied the effect of concentration of Thesit and pH of the medium on the stability and catalytic rate of this lipase towards both butyrate (short tail, water soluble) and oleate (long tail, water insoluble) substrates. Their probes included fluorescence correlation spectroscopy, time-resolved anisotropy decay and temperature-induced unfolding. From these studies, they concluded that the lipase attains its maximum catalytic turnover rate for insoluble long tail oleate substrate when the latter is bound to the detergent micelle at pH 8.5. In this context, it is important to note that the enhancement of lipase activity in the said environment is not necessarily due to any structural changes, as detergent induced structural effects are minimal. 75,76 Herein, our aim is to derive a clear molecular level picture of the evolution of interaction between a lidless lipase and non-ionic detergent with respect to pH and detergent concentration. Towards this aim, atomistic molecular dynamics simulations of BslA-Thesit(C12E8) system in water with varying compositions of detergent at two different pH values have been carried

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out. Anticipating our results, we observe an interesting difference in the location of binding of the detergent molecules on the enzyme, on the pH. The result is an evidence of pH induced rotation of the enzyme at the enzyme-detergent interface, an aspect whose importance was highlighted in the discussion above. The reorientation of the enzyme is explained as due to changes in enzyme surface hydrophobicity as well as in its surface charge redistribution arising from the different protonation states at the two values of pH studied here.

Computational Details 1. System preparation The X-ray crystal structure of substrate-free BslA (PDB ID: 1i6w 51 ), was taken from RCSB protein data bank. 77 1i6w is a homodimer. Thus, two sets of simulations were carried out: of the dimer and of the monomer independently in solution, both sets with varying amounts of surfactants. For the monomer simulations, chain A included in the crystal structure was chosen. Two residues (Ala1 and Glu2) were missing in this structure. The coordinates for atoms in Glu2 were taken from the another representative structure (chain B), whereas the same for Ala1 were generated by PyMOL. 78 The protonation state of each residue in the protein at pH 8.5 and 10 was determined using the ProteinPrepare tool available in PlayMolecule repository. 79 At pH 8.5, the side chain of residues Lys23 (charge: +1) and Tyr139 (charge: 0) are protonated, whereas, they are deprotonated at pH 10 (charge: 0 and -1, respectively). The crystal structure of Thesit detergent (C12E8) was obtained from RCSB ligand explorer. The initial structures for one molecule of this protonated BslA together with different numbers of detergent molecules (0, 1, 5, 15, 30 and 60) were created using PACKMOL A edge length) followed by solvation in water and software 80 (initially in a cubic box of 100 ˚ addition of ion(s) to neutralize the system. Thus, twelve different systems (six each for pH 8.5 and 10) were generated. Additionally, BslA has also been simulated at pH 9.3 (where 6


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the side chain of both Lys23 and Tyr139 are neutral), both in the absence and presence of detergent. We have also simulated BslA in its dimeric form in the presence of 0, 30 and 60 detergent molecules per BslA dimer, each starting from two independent configurations. Details of all the systems and compositions are summarized in Table S1.

2. Simulation run GROMOS54a7 united-atom force field 81 parameters for the protein and detergent were used along with the SPCE model 82 for the water molecules. For all systems, a steepest descent energy minimization was performed keeping position restraints on heavy atoms of the lipase with a force constant of 103 kcal/mol/rad2 . This minimized structure was then equilibrated in multiple steps. Firstly, a 300 ps NVT (at 300 K) followed by a 500 ps NPT equilibration (at 300 K and 1 bar) were carried out with position restraints on the heavy atoms of the enzyme with a force constant of 103 kcal/mol/rad2 . The NPT run was extended further for a duration of 100 ns by removing all position restraints; the last 50 ns of this NPT trajectory was used for all the analyses reported here. The Bussi-Donadio-Parrinello velocity rescaling thermostat 83 with coupling constant 0.5 at 300 K was used in the NVT runs and the Nosé-Hoover thermostat 84,85 and Parrinello-Rahman barostat 86,87 with coupling constant of 1.0 ps for both, at 300 K and 1 bar respectively were used in the NPT runs. An integration time step of 2 fs was used along with LINCS constraints 88 on all bonds. Particle mesh Ewald (PME) method 89 with cutoff distance of 14 ˚ A was used to treat the long-range electrostatic interactions. All systems were simulated using GROMACS 5.1.4. 90–95 Using this protocol, three independent trajectories starting from different initial configurations for each of the twelve systems (containing monomeric enzyme) were produced to have good statistics. Thus, in total, thirty six different MD simulations each of 100 ns have been performed for this study. For details of the system simulated, see Table S1 of SI. In each of the three simulations for each system, the detergent molecules were placed in different, arbitrary initial configurations 7



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with respect to the enzyme. All these initial configurations are presented in Figures S1 to S7 of the SI.

3. Analysis The binding enthalpy for detergents with protein in water was calculated using the following relation:

∆Hbinding = (Vprotein+nDET +water + Vwater ) − (Vprotein+water + VnDET +water )

(1)

where Vprotein+nDET +water is the average potential energy of the system containing protein with n number of detergent molecules in water and the last two terms represent the average potential energies of protein and n DET molecules, respectively, individually in water. For this calculation, systems with 0, 1, 5, 15, 30 and 60 detergent molecules in water in the absence of BslA were prepared and were ran by following the same protocol as described before. Also, while calculating the average potential energy, the number of water molecules present in each system was kept identical. Details of the simulations are in Table S1 of SI. Trajectories were visualized using VMD software. 96 PyMOL software 78 and VMD were used to prepare the graphics. Unless mentioned otherwise, the analyses were performed using tools available in GROMACS 5.1.4 software. The systems are referred herein by their acronyms, e. g., nDET pH8.5 represents the system containing one BslA molecule together with n number of detergent molecules in water. All the results shown here were averaged over the corresponding three independent trajectories for each system.

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Results and Discussion 1. Comparison of structure and activity of BslA at pH 8.5 and 10 1.1 Alternative conformations of catalytic Ser77 Kawasaki et al. 97 determined the X-ray crystal structure of BslA at 1.3 ˚ A resolution and identified two possible conformational states (PDB ID: 1isp). In its active conformation, the side chain oxygen atom (OG) of the catalytic serine residue (S77) forms a hydrogen bond with one of the imidazolium nitrogen (NE2) of another residue H156 from the catalytic triad of BslA, 97 as shown in Figure 2 (left). However, in its inactive conformation, OG forms a hydrogen bond with the imidazolium nitrogen (ND1) of the nearby residue H76. A similar observation was reported for cutinase and acetylxylan estarase as well. 98,99 Like BslA, the latter two enzymes also contain an α/β-hydrolase fold and do not have any profound lid domain. 97–99 Thus, it was concluded by Kawasaki et al. 97 that this flip-flop movement of the catalytic serine residue between active and inactive conformations is a generic feature for estarases (and lipases) containing an α/β-hydrolase fold and lacking a lid domain. In our simulations of BslA in aqueous solution too, both these conformational states are observed. The catalytic S77 flip-flops, with equal probability between the active and inactive states with an average H-bonding distance of 2.8 ˚ A, irrespective of pH (Figure 2 right). Our observation is thus similar to the X-ray crystallographic structure, 97 as both active and inactive conformations of Ser77 are seen. Thus, the present computational model for BslA in water can realistically represent the characteristics of the BslA enzyme.

1.2 Surface area The hydrophobic as well as total surface area of the protein increases with decrease in pH, as shown in Figure 3. This observation is in line with the fluorescence spectroscopic analysis reported in literature, 76 where this fact was clarified by investigating the binding of the chromophoric probe ANS (8-anilino-1-naphthalene-sulfonic acid) with the lipase and also by 9



Figure 2: Left: Active-inactive conformations of oxygen atom OG from the side chain of catalytic residue S77 observed in the X-ray crystal structure of BslA (PDB ID: 1isp). 97 Protein: white transparent cartoon; S77, H156 and H76: licorice with carbon:green, nitrogen:blue and oxygen:red. Right: Distribution of hydrogen donor-acceptor distances employed to identify active and inactive states of S77, for BslA in water in the absence of detergent at pH 8.5 and pH 10.

Figure 3: Mean total, hydrophobic and hydrophilic surface area of BslA at pH 8.5 and 10 at different detergent concentrations. fluorescence quenching of two, single tryptophan mutants W31F and W42F. The exposure of the hydrophobic residues is, in general, more prominent in the presence of detergent (Figure 3). A similar behavior has been reported for several lipases during their interfacial activation, 8–12,36,50 where the hydrophobic residues get gradually exposed to the solvent with decrease in solvent polarity (i. e., with increase in substrate, surfactant or ionic liquid concentration). It is also found that the region around the active site is the most hydrophobic both at pH 8.5 and 10, but its spatial extent is larger at pH 8.5 than at pH 10 (Figure 4).

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Figure 4: Solvent exposure of BslA surface at pH 8.5 and 10, in pure water. Hydrophobicity increases as color changes from white to red. Active site residues are present within the blue circle. Snapshots of the corresponding surfaces are taken at the same orientation (viewed from the top of the head region as discussed later) at both pH. 1.3 Correspondence between substrate-binding and detergent-binding residues Triglycerides, the generic substrates of lipases, 38 are obtained when the three hydroxyl groups of a glycerol molecule are substituted by three same or different ester groups. The (mainly) alkyl and/or acyl tails of these three ester groups form the three side chains of triglyceride: sn-1, sn-2 and sn-3. Through a molecular docking approach, Dijkstra and coworkers identified the binding of C8 -triacylglyceride-inhibitor to the active site of BslA (PDB ID: 1i6w). 51 It was found that while residues L108, L140, A105, M78, I135 and I12 interact with the sn-3 chain, residues I157, L160, Y161, N18, A15, F17, I135, G155 and M134 interact with the sn-1 and sn-2 chain of the triglyceride (Figure 5(a)). The active site catalytic triad (S77, W156 and D133) and the oxyanion hole (I12 and M78) (which stabilizes the oxyanion intermediate generated from the catalytic reaction) interact with the glycerol part of the triglyceride 51 (Figure 5(a)). From Figure 5(b), it is observed that most of these residues (except 105 and 108, and also residues within the head region, except 102-110 and some of 75-90 and 130-140) possess higher RMSF at pH 8.5 than at pH 10. The higher flexibility of these residues suggests that they are more accessible to the substrate, surfactant and solvent. This fact is further

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confirmed from a comparison of the solvent accessible surface area (SASA) of residues of the head region (Figure 5(c)). An overlay of the lipase structure at pH 8.5 and 10 (Figure 5(d)) too confirms that these residues are the main ones interacting with either the substrate or the monomeric surfactant.

1.4 BslA: having a partial lid? Eggert et al. 100 found that exchanging the loops consisting of residues 39-51 with an extrinsic lid sequence reduced the activity of BslA. From this observation, Kubler et al. 76 raised a question that whether residues 39-51 can serve as lid for this enzyme. From our simulations, although these residues are found to be more flexible at pH 8.5 than at pH 10 (Figure 5(b)), they are not found to cover (close) the active site in any of the systems (at both pH 8.5 and 10). Thus MD simulations do not offer any evidence for this conjecture. On the other hand, our simulations suggest that the short α-helix consisting of residues 154-163 can possibly serve as a partial lid in BslA. In the absence of the detergent, at pH 8.5, this short helix is far away from residue S77, making the lid to be in the ’open’ state (Figure 6 (a) and (b)). Upon increase of pH from 8.5 to 10, this short helix moves towards the catalytic residues S77 and partially covers the latter (Figure 6 (a) and (b)). Thus, depending on pH, this helix partially covers (or uncovers) the catalytic residue. But, in the presence of the detergent and at pH 10, this helix moves away from the active site and remains almost at the same distance away from residue S77 as it was in pH 8.5 (Figure 6 (b)). Thus, the presence of detergent can convert the enzyme from its inactive closed form (covered active site) to its active open form (open active site), which is essentially the signature of interfacial activation. 8–12 Furthermore, the residues G155, H156, I157, L160 and Y161 from this helix have already been recognized as substrate-binding residues (Figure 5 (a)). As a result, in the closed state of the enzyme, this helix can bind with the substrate present in the active site cavity. Thus, by considering all these aspects, we feel that this short amphiphilic α-helix can serve as a partial lid for BslA.

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Figure 5: Head region around BslA active site. (a) Interaction between substrate-binding residues of BslA (PDB ID: 1i6w) and C8 -triacylglyceride-inhibitor. 51 Reprinted with permission from Elsevier. (b)RMSF (root mean square fluctuation) averaged over each residue with respect to the equilibrated structure of the enzyme at pH 8.5 and 10. Residues corresponding to the head region (consisting of residues 10-20, 40-55, 75-90, 102-110, 130-140 and 150-165) are highlighted. (c) Solvent accessible surface area (SASA) of head region at pH 8.5 and 10 calculated over 50-100 ns of corresponding simulation trajectories. (d) Similar interactions as depicted in (a), are observed in our simulations of BslA with one Thesit molecule at pH 8.5. Here BslA, head region and detergent are shown in white and yellow transparent cartoon, and licorice (with carbon atoms as green and oxygen atoms as red) representations, respectively. Substrate-binding residues are also represented as licorice. Active site residues; residues that were supposed to interact with sn-2 (also sn-1), and sn-3 of model substrate triglyceride 38,51 are labeled with text color red, green and blue, respectively. A correspondence between triglyceride-lipase interaction as seen in (a) and Thesit-lipase interaction as shown in (d) is thus drawn.

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Figure 6: A short α-helix can serve as a partial lid of BslA. (a) Position of the α-helix backbone with respect to catalytic residue S77, in the absence of the detergent at different pH. The helix is represented as cartoon with color green (pH 8.5), cyan (pH 9.3) and magenta (pH 10). The whole protein is represented as white transparent cartoon. (b) Distance between the center of mass of the helix and the Cα residue of S77 with respect to 50-100ns simulation trajectory, for 0DET and 30DET systems at different pH. Thus, from the discussion so far, we can conclude that as a consequence of having higher hydrophobic surface area (especially around the active site region, termed here as ”head” region) at pH 8.5, BslA is catalytically more active at this pH than at pH 10. This is in line with the experimental results on BslA by Kubler and coworkers. 76 In the rest of the discussion, we focus on the correlation between solvent accessible hydrophobic surface area and activity of BslA in the presence of the detergent, Thesit.

2. Effect of detergent on enzyme at pH 8.5 and 10 From the RMSD and radius of gyration (Rg ) of the protein backbone both at pH 8.5 and 10 (Figure S8), it is clear that in the systems with protein to detergent ratio ranging from 1:0 to 1:30, the enzyme’s structure is intact, whereas the system with 1:60 ratio is quite different from the rest. Also, in terms of protein-detergent relative orientation, the system with 1:60 ratio does not differ from that at 1:30 ratio, at both pH 8.5 and 10 (Figure S9). Thus, for the rest of the discussion, we will not discuss the 1:60 system.

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2.1 Qualitative validation of the enzyme to detergent ratio A tractable system size for molecular dynamics simulation at atomistic level is one where the simulation box length is around 10 nm. With such a limitation, it is not possible to reproduce the behavior of an aqueous macromolecular solution quantitatively. Thus, we can only predict the protein-detergent interaction from a molecular perspective and its effects on the enzyme, but we can neither estimate the critical micellar concentration (CMC) of the model detergent, nor the micelle-enzyme interaction. However, a qualitative view can certainly be obtained. In this regard, it is necessary to qualitatively validate the present protein to detergent ratio with the experimental one 75 (where the concentration of protein and detergent are of the order of nM and µM respectively, i. e., protein:detergent :: 1:1000). The van’t Hoff enthalpy of BslA unfolding in aqueous Thesit environment, ∆H0 unf (generally endothermic), was calculated by Kubler et al. 75 by analyzing the tryptophan (W31 and W42) fluorescence intensity. They observed that ∆H0 unf for BslA induced by detergent at pH 8.5 is lesser than at pH 10, for Thesit concentration below CMC (100 micromolar). Consistent with experiments, the BslA-Thesit binding enthalpy (generally exothermic), calculated from MD simulations is also found to be lesser at pH 8.5 than at pH 10 (Figure S10). The average binding enthalpy for different number of detergent molecules with protein in water is comparable to the average energy required for the detergent induced unfolding of protein in water. 75 Thus, the behavior of binding enthalpy obtained from simulations with pH and on detergent concentration is in reasonable agreement with the experimental results. 75 Thus, we believe that the simulated systems with enzyme to detergent composition (from 1:0 to 1:30) represent experimental conditions and can be used to explain the qualitative features of BslA-detergent interactions. At pH 10, the binding enthalpy is considerably smaller than at pH 8.5; for 15 and 30 detergent molecules it is almost endothermic, the reason for this is explained in terms of the hydrophobic SASA and the change in the binding regions of the detergent with concentration at pH 10, in Figure S10. 15



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2.2 Detergent-interaction sites of BslA depending on BslA-detergent composition As observed by Kubler et al., 75 in the monomeric form, the detergent molecule, irrespective of pH, interacts mainly with the residues near the active site (’head’ region); i. e., substrate-binding residues, especially with those that are expected to bind with the sn-3 chain of the model substrate triglyceride, as shown in Figure S11. With increase in detergent concentration, most of the detergent molecules get attached to the ’head’ region in oligomeric form, especially to the sn-3 chain binding region, irrespective of pH, as shown in Figure S12 for systems containing five detergent molecules. However, some of the detergent molecule(s) at pH 8.5 attach(es) to the region opposite to the head region of the lipase, termed as the ’tail’ region. Whereas at pH 10, some of the detergent molecule(s) not bound to the ’head’ region, attach to the lateral region (side region of the lipase surface in between the ’head’ and ’tail’ regions). Thus, with the increase of detergent concentration (i.e., as the possibility of detergent oligomer formation increases), few changes depending on pH are observed. This behavior gains prominence with a further increase of detergent concentration (protein:detergent :: 1:15) (Figure S13). Surprisingly, at even higher detergent concentration (i. e., protein:detergent :: 1:30), the lipase-detergent relative orientation becomes pH-dependent. At pH 8.5, the detergent oligomer attaches mainly to the head region (and sometimes to both the head and tail regions with larger detergent population at the head region) and partially (Figure 7 (b)) or fully (Figure 7(a) and (c)) covering the active site. On the other hand, at pH 10, the detergent oligomer attaches mainly to one portion of the side (lateral) region (consisting of two α-helices α1 (residues 16-28) and α2 (residues 48-66), and one β-sheet β2 (residues 35-42), and their connecting loops); and sometimes to both this α1-β2-α2 region (Figure 1) and the lateral region on its opposite side, with a larger detergent population at the former (Figure 7 (d), (e) and (f)). Thus, at low ratios of detergent to lipase, the former attaches to the head region of the enzyme, irrespective of pH. At higher concentrations, the detergents start to bind to both 16


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Figure 7: Snapshots from three independent MD simulations of the system with enzyme and thirty detergent molecules in water at pH 8.5 and at pH 10. BslA is shown with the same view as shown in Figure 1(a). Here BslA, head region and detergent are shown in white cartoon, yellow cartoon and licorice (with C12 tail as cyan and E8 head as red) representations, respectively. Active site residues are represented in van der Waals spheres. the head (mainly) and tail regions of the lipase at pH 8.5, and to the side region (mainly at the α1-β2-α2 region) of the lipase at pH 10. So, with increase in detergent concentration, pH becomes a controlling factor behind lipase-detergent relative orientation. Although the crystal structure of BslA (1i6w) is in a dimeric form, gel filtration and dynamic light-scattering (DLS) experiments indicate that it is unlikely to exist as a functional dimer; 51 in particular, at pH 10, DLS experiments showed only a monomeric enzyme. 51 Furthermore, the two chains in the dimer (as seen in the crystal structure) interact via their respective tail regions, exposing the respective active sites (present in their head regions) to the solvent. In the crystal structure, the side chain of Glu2 from the tail region of Chain B is hydrogen bonded to Lys170 and Asn174 present in the tail region of Chain A. 51 Results of the BslA-detergent simulations in the dimeric form of BslA are presented in Figures S14, S15 and 17



S16 for various detergent concentrations (the initial configurations of detergents around the dimer for the latter two runs are presented in Figures S6 and S7 respectively). The distance between the two subunits of the dimer at all detergent concentrations irrespective of pH, is close to the distance found in the crystal structure, implying the stability of the dimer within MD simulation timescales. The nature of detergent binding to the dimer is quite similar to that in the monomer. In the dimer, the detergent aggregates bind to the head region of each of the subunits at pH 8.5 and to their side regions at pH 10. As the two subunits in the dimer interact via their tail regions, the detergent binding to the dimer proceeds in the same fashion as it would in a monomer, i.e., to the head and side regions which are distal to the oligomeric interface. Note that the comparison between monomeric and dimeric simulations should be done at the same ratio of BslA to detergent. Thus, 15DET simulations of the monomer should be compared to 30DET simulations of the dimer. As the results from the simulations of the dimeric form of the enzyme mirror those from the monomeric ones, the rest of the discussion is exclusively devoted to simulations of the monomeric form.

2.3 pH-induced rotation of BslA at lipase-detergent interface We note that the BslA (PDB ID: 1i6w) conformation at pH 8.5 is comparable to the Lout conformation of BslA 56 (PDB ID: 4bhu) discussed earlier, while the conformation of the simulated enzyme at pH 10 is comparable to the Lin conformation (Figure 8). The head region of BslA in our simulations is also similar to the aforesaid hydrophobic cap (Figure 8). The nature of lipases is such that they prefer to reside near the aqueous-lipidophilic interface of a water-lipid biphasic system. 8–12,36,50 Thus, the present investigation strongly suggests a similar kind of rotation (as observed by Brandani et al. 56 ) of BslA (1i6w) near the lipase-detergent interface on increasing pH from 8.5 to 10 (Figure 11), as discussed later. A pH-induced reorientation of (fungal) hydrophobin at the air-water interface has recently been observed using sum-frequency generation spectroscopy, 101 which is very similar to the one seen in the bacterial hydrophobin BslA, reported here.

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Figure 8: Comparison between two different BslA enzymes with PDB IDs 1i6w and 4bhu. Top: Two forms of BslA (1i6w) at two different pH, in water in the absence of detergent obtained from the present simulations. Bottom: Two forms of BslA found in the crystal structure (PDB ID: 4bhu 55 ) and termed as Lout and Lin by Brandani et al. 56 Enzyme: white cartoon; head region (or hydrophobic cap): yellow cartoon; partial lid (residues 154-163): green cartoon; residues (H10, K44, M78, A81, H156, L160, Y161) passing through highest conformational transition: licorice. Right: The same set of residues also exhibit significant change in SASA between pH 8.5 and 10 (or Lout and Lin conformations) plotted here for both 0DET and 30DET systems. At this point, it is interesting to note that the residues (M78, H156, L160 and Y161) which distinguish between the Lin and Lout conformers (Figure 8) are the ones which are found to take part in substrate-binding (Figure 5 (a)); and residues H156, L160 and Y161 are the parts of a short α-helix which can act as a partial lid for this lipase (Figure 6). Furthermore, the Lout and Lin conformations are seen to be stable at pH 8.5 and pH 10 respectively, even in the presence of detergents, as can be understood from the SASA values for the residues involved (right panels of Figure 8).

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2.4 pH-induced rotation of BslA at interface: rationalization C12E8 detergent was found to interact with proteins by forming small roughly spherical micelles. 71,73,102 In the present study, at sufficient numbers (15 or more), they are found to form aggregates in which the C12 hydrophobic tail is largely buried inside (the micelle-like aggregate) and the polyoxyethylene head is exposed outside (Figures S13 and 7). Thus, the interaction of BslA with detergent aggregates happens mainly through the amphiphilic polyoxyethylene head. As a result, both electrostatic and van der Waals mode of BslA-detergent interaction become important. 72 This fact is supported by the observation that the Lin/out residue group possesses both charged (K44) as well as neutral (H10, M78, A81, H156, L160, Y161) residues (Figure 8). Although slightly polar polyoxyethylene oxygen and polar hydroxyl oxygen are present in the head group of the detergent, the average number of BslA-detergent hydrogen bonds are few (only around 3 to 4) (Figure S17). This observation suggests that even though BslA interacts mainly with amphiphilic head group of detergent, the mode of this interaction is mostly of van der Waals type. Rationalization I: The interfacial activation of lipases is mostly related to the van der Waals part of BslA-detergent interactions. 8–12,36,50 Furthermore, the binding of C12E8 detergent to proteins was reported in literature to be a measure of the hydrophobic surface area of the latter. 71,73 The hydrophobic SASA of the head region in the simulated BslA here, is larger than that for the side region at pH 8.5, whereas it is the other way around at pH 10 (Figure 9). As a result, detergent molecules get attached to the head region at pH 8.5 and to the side region at pH 10 (Figure 7). Further discussion on the contribution from electrostatic and van deer Waals counterpart on protein-detergent binding as well as the effect of hydrophobic and hydrophilic parts of SASA of BslA on lipase-detergent interaction can be found in Supporting Information. Rationalization II: The pH-induced BslA rotation at the interface can also be rationalized in terms of the electrostatic potential (ESP) map of the lipase surface (Figure 10). With the increase of pH from 8.5 to 10, the side chain of residue Tyr139 from the head region gets 20


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Figure 9: Hydrophobic contribution to the SASA of head and major side regions with respect to number of detergent molecules at pH 8.5 and 10. deprotonated and becomes negatively charged. This changes the ESP of the neighboring region (within the black circle) from mostly neutral to positive which in turn converts the ESP of the region nearby (within the magenta circle) from positive to negative. Similarly, the positively charged side chain of residue Lys23 from the major side (lateral) region (α1-β2-α2 region) gets deprotonated and becomes neutral. This, in turn, changes the ESP of the neighboring region (within the black circle) from negative to mostly neutral. We have already observed that the lipase-detergent interaction is expected to be generally van der Waals type, though electrostatic interactions are also important. Thus, the detergent would prefer to attach to the protein surface having either neutral or a slightly positive ESP. This causes the detergent aggregates to move from the head to the major side (lateral) region upon increase of the pH from 8.5 to 10; in other words, a pH-induced rotation of BslA occurs near the BslA-detergent interface.

2.5 pH-induced rotation of BslA can control its catalytic activity Upon decreasing the pH from 10 to 8.5, the lipase rotation permits the active site (i.e., the head region) to move towards the detergent phase of the lipase-detergent interface. The outcome of this pH-induced rotation is two fold. First, the closeness of lipase active site to 21



Figure 10: Electrostatic potential (ESP) map for head and major side (lateral) regions of BslA, for systems containing 15 and 30 detergent molecules at both pH. ESP is scaled to the range (-1,+1) Volt. The residues Lys23 and Tyr139, whose side chain get deprotonated with increasing pH from 8.5 to 10, are represented as green color sticks. Detergent molecules are represented as lines with carbons as yellow and oxygen as red colors. Black and magenta circles show the significant changes due to change in pH.

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the lipase-detergent interface (Figure 11 (a) and (b)) can enhance the interfacial activation of this lipase. Secondly, the substrate of lipases being lipidophilic, are predominantly sparingly soluble or insoluble in water. So, substrates such as long chain triglycerides or 4-MU oleate prefer to be bound to detergent oligomer or micelle. Thus, at pH 8.5, the proximity of the active site to the detergent phase facilitates the substrate to come in close to the active site (Figure 11 (b) and (c)). This makes BslA to be most catalytically active at pH 8.5. On the other hand, as detergent aggregates (or micelles) are not able to approach the active site at pH 10, BslA is less active (Figure 11 (b) and (c)). Kubler et al. 75 has enunciated that BslA at pH 8.5 shows its highest catalytic turnover rate towards micelle-bound substrates, whereas, at pH 10, it shows highest catalytic turnover rate towards free substrates. A similar scenario has been observed in the present study (Figure 11 (b)) due to the closeness of active site to the lipase-detergent interface at pH 8.5 compared to pH 10. In this manner, the partial or full blocking of the active site by detergents at pH 8.5 (Figure 7) is overcome. All these results tie in neatly with the experimental observations. 75,76

2.6 pH-induced rotation of BslA and the bioavailability of BslA-coated drugs We conjecture briefly on a possible utility of the pH-induced rotation of BslA here. With the increase in pH from 8.5 to 10, the movement of head region of BslA away from the interface is akin to the separation of peripheral membrane proteins from the membrane with the enhancement in pH. 103 Some of these peripheral membrane proteins are hydrolases, catalysing the hydrolysis reaction. BslA (containing α/β-hydrolase fold) too catalyses the hydrolysis of lipids and thus, like phospholipases, shows similar characteristics as peripheral membrane proteins. Independently, Bimbo et al. 69 found the dissociation of (fungal) hydrophobin coating over porous silicon microparticles (a widely used drug carrier 74,104–116 ) with increase in pH. Combining these two observations, it can be said that the biofilm coat formed by BslA self-aggregation (see Introduction) around drug carrier nanomaterials can be prone to be temporally attached to the cell membrane. Thus, pH-induced rotation of BslA can be

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Figure 11: (a) Effect of pH change on protein-detergent relative orientation, shown in the presence of thirty detergent molecules at different pH. Graphical representation and color scheme are similar to Figure 7. Change in charge on the side chain of the respective residues undergoing protonation/deprotonation during the change in pH, are also mentioned. The same scenario in presence of substrate has been shown as schematic (b). This change in BslA-detergent relative orientation can be compared to the rotation of BslA at lipid-water interface as shown in the schematic representation (c). Graphics and Color scheme: water: sky blue; monomeric detergent: deep green; detergent micelle: pale green circle; hydrophobic head region of BslA containing active site: red; hydrophilic region of BslA: deep blue; substrate: pink. White asterisk represents the position of the active site of BslA. In (b), pale green circle containing pink zigzag pattern represents micelle-bound substrate. At pH 10, the substrate is free, whereas at pH 8.5, it is bound to the lipid phase (or detergent aggregate) as shown in (c). 24 ACS Paragon Plus Environment

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related to the bioavailability of BslA coated drugs. This is supported by the fact that BslA is found to form stable oil-in-water microcapsules. 117,118 However, these conjectures need to be substantiated by both MD simulations and experiments particularly using local probes.

Summary and Conclusions Like other lipases, 55,56 BslA exposes its hydrophobic residues in its most active state at pH 8.5. BslA has more hydrophobic surface area at this pH, which is located mainly around the active site (head region). Based on simulation results at pH 8.5 and pH 10, we suggest that the short amphiphilic α-helix (formed by residues 154-163) can serve as a partial lid for BslA. Although monomeric detegents, irrespective of pH, interact with the lipase through the substrate binding residues of the head region, the interaction of oligomeric detergents at high detergent concentration becomes controlled by pH. At pH 8.5, detergent oligomers interact through the head (and sometimes tail also) region, whereas at pH 10, they interact with the lateral region of the lipase.

This pH-dependent lipase-detergent orientational

preference triggers the rotation of the lipase near the water-lipid interface. This makes BslA to be more active at pH 8.5 and rationalizes its highest catalytic turnover rate towards micelle-bound substrates, as reported experimentally 75 and less active at pH 10 and exposes its highest catalytic turnover rate towards free substrates. 75 All these observations from the present computational study with detailed molecular perspective reproduce nicely the previous computational and experimental findings reported in literature 51,56,75,76 and offer microscopic insights to the same. The chief results which have emerged from the current study are: • (i) a correspondence between residues which bind to the substrate 51 and those to the monomeric detergent has been established • (ii) the short α-helix consisting of residues 154-163 which show a transformation between open and closed states with pH change can act as a partial lid 25



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• (iii) the hydrophobic surface area of BslA is higher at pH 8.5 than at pH 10 in line with experiments; 76 and the specific contribution to it from the residues near the active site to the same has been demonstrated • (iv) a novel pH-induced rotation of bacterial hydrophobin BslA at lipase-detergent interface 56 (similar to the experimental observation for fungal hydrophobin at the air-water interface 101 ) has been observed. The same provides a rationale for the experimentally observed increase in enzymatic activity at pH 8.5 over that at pH 10. 75 Other than the pH-dependent rotation of BslA, the enhancement of aqueous-lipidophilic interfacial diffusion of BslA in the presence of detergent may also underlie the detergent-induced increased catalytic activity, as conjectured by Kubler et al. 75 For a large macromolecular entity like the BslA enzyme, both the rotation near and diffusion through aqueous-lipidophilic interface would be slow processes. So, a proper investigation of the dynamics of these slow events demands molecular simulations at coarse-grained level (together with enhanced sampling techniques), 119,120 which is in progress in our group. Most of the solvent accessible hydrophobic residues of globular protein 51 BslA are situated around the active site (head region), and rest of the BslA surface is largely hydrophilic. This makes BslA a Janus particle. 56 Due to the pH-induced rotation, this lipase can be used as a switchable Janus nanocolloid for several biotechnology applications, especially in the field of targeted drug delivery where the biofilm coat 121 formed by self-aggregation of BslA over drug carrier increases the biocompatibility and bioavailability of lipophilic drugs. In this regard, further investigations are needed to improve this switchability by adjusting external parameters and/or by fine-tuning of protein’s local chemical environment. It would also be interesting to find out whether the two important properties of BslA, i. e., rotation near aqueous-lipidophilic interface and self-aggregation are related or not.

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ASSOCIATED CONTENT Supporting Information Supporting Information contains details of the system preparation along with initial configuration for every systems; RMSD and Rg of all the system at pH 8.5 and 10 with respect to simulation time; a comparison of BslA-detergent relative orientation between 30DET and 60DET systems at pH 8.5 and 10; average protein-detergent binding enthalpy; BslA-detergent relative orientation for 1, 5 and 15 detergent molecule(s); distance between two subunits of BslA in its dimeric form; BslA-detergent relative orientation for 30 and 60 detergent molecule(s) for dimeric BslA; number of BslA-detergent hydrogen bonds with respect to 50-100ns simulation trjectory for 30DET system at pH 8.5 and 10; total, hydrophobic and hydrophilic SASA of head and major side regions with respect to number of detergent molecules at pH 8.5 and 10; and the electrostatics and van der Waals counterparts of average BslA-detergent binding enthalpy with respect to detergent concentration at pH 8.5 and 10. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] +9180 22082808

Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENT We acknowledge the Department of Science and Technology, India for support. SD acknowledges Council of Scientific and Industrial Research for partial financial support.

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pH-Induced Rotation of Lidless Lipase LipA from Bacillus subtilis at

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