Self-Assembly Nanostructures of TriglycerideâWater Interfaces

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Self-Assembly Nanostructures of Triglyceride−Water Interfaces Determine Functional Conformations of Candida antarctica Lipase B Sven P. Benson and Jürgen Pleiss* Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D - 70569 Stuttgart, Germany S Supporting Information *

ABSTRACT: Candida antarctica lipase B (CalB) acts as a lipase when adsorbed to an acylglyceride interface and as an esterase when exposed to an aqueous environment. The effect of the molecular self-assembly nanostructure of triglyceride−water interfaces on structural conformations of adsorbed CalB and the implications to its catalytic function were studied by molecular dynamics simulations. Systems of CalB adsorbed to interfaces and solvated in water were compared. The two environments induced relative motions of helices α5 and α10 that resulted in open and closed conformations. The open conformation was stabilized by interactions between the polar and nonpolar amino acids of α5 and α10 and the nanostructure of triglyceride aggregates, which self-assembled into crystalline-like patterns of alternating polar and nonpolar lamellae. Thus, the structure of CalB has been adapted by evolution to the geometric constraints imposed by the interface nanostructure for optimized catalytic activity. Helices α5 and α10 have two functions. As mobile elements, they ensure access of bulky substrates to the active site in the open conformation. As a part of the active site pocket, they ensure binding of substrate molecules in a productive orientation near the active site. In water, access to the binding site is limited, and the smaller substrate binding site is beneficial for the binding of small, water-soluble substrates. The CalB crystal structure commonly used for protein engineering studies represents an intermediate state between open and closed, and may thus not be adequate to assess the function of CalB, neither as lipase nor as esterase.

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complex lid designs have been reported that involve concerted rearrangements of secondary structure elements, e.g., in Candida rugosa lipase.9 Candida antarctica lipase B lacks a clearly distinguishable lid structure10,11 but features two flexible α-helices, α5 and α10, that flank the active site entrance. Particularly, α5 was already proposed as a potential lid candidate in the first report on a CalB crystal structure, due to its proximity to the active site entrance.12 Since then, the high flexibility and conformational variability of α5 were demonstrated in multiple molecular dynamics studies of CalB in solution,13−19 and hence, α5 was discussed as a putative lid.15,18,20 Whether CalB shows interfacial activation is under discussion; it was refuted in early studies,10,21 found to be indistinct in the meantime,22,23 and was only recently reported for a bulky substrate.13 This apparent substrate dependency supports the notion that lid structures and the existence of open conformations may not be a sufficient criterion to mechanistically explain interfacial activation but that it is instead determined by a convolution of properties pertaining to the dielectric environment.24 Substrate-dependent interfacial activation is not restricted to CalB and has been demonstrated for other lipases, e.g., Staphylococcus hyicus lipase.25 Some

ue to the limited water solubility of most industrially relevant substrates, biocatalytic processes in aqueous solvents involve enzymes that are active at substrate−water interfaces formed in suspensions, emulsions, or microemulsions. Lipases are ubiquitous surface-active enzymes that catalyze the hydrolysis of oils and fats under aqueous conditions, e.g., in food processing or waste treatment.1 If transferred to nonconventional solvents, lipases display remarkable stability and are viable catalysts for synthetic esterification or transesterification reactions.2 Lipases differ from esterases by their enhanced activity toward self-assemblies of long-chain acylglycerides, whereas esterases prefer small water-soluble substrates. Lipases are thus hydrolases that have evolved to function most effectively at oil−water interfaces. This phenomenon is commonly referred to as interfacial activation3 and has been attributed to conformational changes of mobile lid structures that are sensitive to different dielectric environments. In water, the lid is supposed to be closed, resulting in an inactive lipase, while the open conformation is induced by adsorption to a hydrophobic interface, providing substrate access to the active site of the enzyme. Recent studies have revealed a direct correlation between interfacial activation and the presence of a lid by comparing wild type Candida antarctica lipase A to truncated lid variants.4 The first lid structures were reported for Rhizomucor miehei lipase5 and human pancreatic lipase.6 Both proteins feature a hinge-like helix flanked by flexible loop regions.7,8 More © XXXX American Chemical Society

Received: December 21, 2016 Revised: March 7, 2017 Published: March 8, 2017 A

DOI: 10.1021/acs.langmuir.6b04570 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. (A) Triglyceride layers were generated from random mixtures of water (blue) and triglyceride (yellow). (B) The systems were equilibrated by molecular dynamics simulations into self-assembled layers. (C) A CalB protein (gray) was attached to the triglyceride layer with the entrance of the active site facing toward the interface to monitor conformational changes (attached state, ATT). (D) After equilibration, the protein was detached from the layers and resolvated in water (detached state, DET) to monitor conformational adjustment to the aqueous environment.

Figure 2. (A) The active site entrance was monitored by the distance rα5α10 between the centers of mass of the Cα atoms of helices α5 (green) and the C-terminal part of α10 (blue). (B) Conformational changes of the active site pocket were monitored by distances between the oxygen atom Oγ of catalytic S105 and Cα atoms of amino acids within a spherical cutoff of 10 Å, as well as Cα atoms of amino acids corresponding to the acyl (red), medium (yellow), and large (orange) substrate binding pockets.14 (C) Distances rOγCγ, rCγCα, and rCγNζ between residues D145 and T158 as well as D145 and K290 were analyzed as potential candidates for salt bridge stabilization of an open state in accordance with a crystal structure by Uppenberg (PDB: 1TCA12) and compared to (D) a closed state observed in the crystal structure by Stauch et al. (PDB: 5A6V24).

first time demonstrated the existence of CalB closed conformations that occur due to considerable unfolding of α5 in one of the chains in a high-resolution CalB dimer crystal structure. In light of these recent reports, we have conducted an indepth molecular dynamics study on CalB adsorption onto triglyceride−water interfaces in full atomistic detail, where we have investigated determinants of interfacial activation, with a particular emphasis on the interplay between CalB structure and the nanostructure of the triglyceride aggregate.

lipases, in fact, do not display any interfacial activation at all, such as guinea pig and coypu lipases,26 despite sizable lids that are structurally homologous to pancreatic lipase, which in turn displays enhanced activity upon interfacial adsorption. A more comprehensive perspective on interfacial activation also includes the dynamics and conformations of substrate molecules at the oil−water interface. This concept relating interfacial activation to the interfacial nanostructure was vaguely defined as “interfacial quality” and incorporates factors such as hydration states, increased substrate availability, or preferential substrate orientation during protein adsorption.27 The exact determinants of interfacial activation, however, have yet to be established. CalB is an ideal model system to investigate these interactions, since its lid mechanism is under discussion, and since it acts both as an esterase toward water-soluble substrates and as a lipase toward self-assembled substrate aggregates. Zisis et al.13 recently emphasized the influence of CalB α5 and α10 conformational variability on interfacial activation by computing free energies of binding in conjunction with continuum solvent models. Another recent study by Stauch et al.,24 although not specifically addressing interfacial activation, for the

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RESULTS AND DISCUSSION Interfacial activation describes the correlation between lipase adsorption onto self-assembled substrate interfaces in oil−water emulsions and the enhancement of catalytic activity. Lipases are popular biocatalysts with high optimization potential toward synthetic substrates, nonconventional solvent conditions, and immobilization preparations. However, interfacial activation introduces an additional degree of complexity that makes the rational engineering of lipases particularly challenging. The concept of interfacial activation not only requires an in-depth functional understanding of the biocatalyst itself but also of its B


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Figure 3. Distance rα5α10 between the center of mass of Cα atoms of CalB helices α5 and α10 during 1 μs NPT simulations. CalB was (A) attached (cf. Figure 1C) to a triglyceride layer or (B) detached (cf. Figure 1D) from the layer and resolvated in water. The layers consist of saturated triglycerides with varying acyl chain length: tributyrin (C4, red), tricaproin (C6, green), and tricaprylin (C8, blue). Distance intervals indicate the conformational states of the active site entrance cleft (closed, intermediate, open).

Figure 4. Normalized distribution of distance rα5α10 (cf. Figure 3) of CalB in (A) the attached and (B) the detached state. Triglyceride layers consist of tributyrin (C4, red), tricaproin (C6, green), or tricaprylin (C8, blue).

orientation, since the helices flank the active site entrance and thus directly interact with the substrate aggregate. To measure CalB open and closed conformations during adsorption, the center of mass (COM) distances rα5α10 between helices α5 and α10 were calculated (Figure 2A). The active site entrance cleft was defined as closed, intermediate, or open for rα5α10 < 1.5 nm, 1.5 < rα5α10 < 2.0 nm, and rα5α10 > 2.0 nm, respectively, in agreement with the study of Zisis et al.13 These definitions were also in agreement with measurements conducted on CalB crystal structures12,20,24,28,29 (Table S2) of open, intermediate, and closed conformations (Figure S6A). In six independent MD simulations, the distance rα5α10 varied between 1.0 and 2.5 nm, indicating a wide distribution of accessible α5 and α10 conformations between closed, intermediate, and open states (Figure 3). Within 1 μs, all simulations modeling CalB attached to the triglyceride−water interface (CalB-C4-ATT, CalB-C6-ATT, CalB-C8-ATT) converged to an open conformation with rα5α10 > 2.0 nm (Figure 3A, Figure 4A). Rapid fluctuations of Δrα5α10 ≈ 0.2 nm observed on a ns time scale correspond to local changes in helical secondary structure. Distance variations beyond this fluctuation (Δrα5α10 > 0.2 nm) represent significant conformational changes with respect to the opening or closing of the CalB active site entrance. These concerted motions were predominantly observed on a 100 ns time scale; the gradual convergence from distance rα5α10 = 1.5 nm (closed) to rα5α10 = 2.0 nm (open) for the tricaprylin (C8) system during 1 μs of simulation is representative for this slow conformational shift. However, the opening and closing of the active site entrance cleft can also occur rapidly, as demonstrated by the fast

complex interactions with the solvent environment and the selfassembly nanostructure of the substrate aggregate. Therefore, rational lipase engineering efforts require a comprehensive perspective on all components of the interface, to ascertain enzyme conformations that adequately represent conditions of optimal catalytic activity. To the best of our knowledge, this is the first study that delivers insights on the influence of the substrate aggregate on enzyme structure and function in the context of interfacial activation. We have investigated CalB adsorption with a holistic perspective on all components of interfacial nanostructure by molecular dynamics simulations, modeling the self-assembled substrate aggregate in full atomistic detail. A total of six 1 μs simulations of CalB were performed in conjunction with triglyceride layers consisting of tributyrin (C4), tricaproin (C6), and tricaprylin (C8). Three simulations thereby modeled CalB attached to the respective triglyceride layers (Figure 1C: CalB-C4-ATT, CalB-C6-ATT, CalB-C8-ATT), and another three simulations modeled CalB detached from the interfaces in its adsorbed conformation and then resolvated in water (Figure 1D: CalB-C4-DET, CalB-C6DET, CalB-C8-DET). Thus, two environments typical to lipase and esterase activity were compared, and active site accessibility and active site morphology were analyzed by geometric descriptors. Particular emphasis was placed on the influence of the triglyceride aggregate nanostructure on the corresponding CalB conformations by a detailed moiety association bias analysis.38 CalB Active Site Accessibility Facilitated by Conformations of Helices α5 and α10. Conformational changes of the two helices α5 and α10 are of particular relevance to substrate accessibility during CalB adsorption in a productive C


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Figure 5. Distances were calculated between catalytic residue S105(Cα) and active site Cα atoms to evaluate conformational changes within the substrate binding pocket during adsorption to a triglyceride−water interface. The distance deviation was calculated between simulated systems and the average coordinates of equilibrated CalB (PDB: 1TCA)12 and evaluated for medium (left to right: residues 39, 42, 47, 104, and 225), large (residues 141, 144, 154, 285, 289, and 290), and acyl (residues 134, 138, 157, 189, and 190) pockets. Systems of CalB (A) attached to and (B) detached from triglyceride interfaces of tributyrin (C4, red), tricaproin (C6, green), or tricaprylin (C8, blue) were compared. An independent 1 μs simulation of CalB in water served as a reference starting directly from a crystal structure (black horizontal lines).

expected to be predominantly hydrophobic. Indeed, helix α5 consists of hydrophobic amino acids 139−147 (VLAGPLDAL) except for the hydrophilic D145. Likewise, the C-terminal part of helix α10 consists of hydrophobic amino acids 277−288 (LLAPAAAAIVAG) followed by K290. Residues D145 and K290 were recently proposed as salt bridge candidates by Stauch et al.,24 wherein a CalB closed crystal structure was reported with helix α5 shielding the active site entrance channel from the solvent environment (Figure 2D). Another salt bridge between D145 and T158 was proposed as a stabilizer for the open conformation (Figure 2C). We compared the salt bridge distances from the respective crystal structures with the corresponding distances derived from our simulations (Table S2, Figure S3) and found no evidence to suggest stable salt bridge formation in the open or closed conformations in any of the simulated systems (Figure S4). While the orientation of D145 in crystal structures 5A6V and 5A71 by Stauch et al. implies the existence of a stabilizing salt bridge in the closed state, the orientations of the D145 side chain in our simulations were random, even in distinctively closed conformations of CalB in aqueous solution simulations (Figure S6). The hydrophilic amino acids of α5 and α10 appear to preferentially interact with the aqueous environment. CalB Adsorption Induces Conformational Changes in the Substrate Binding Site. Another potential determinant of enzyme kinetics is the stabilization of a bound substrate molecule near the active site mediated by three binding pockets, the acyl pocket (residues 134, 138, 157, 189, 190), the medium pocket (residues 39, 42, 47, 104, 225), and the large pocket (residues 141, 144, 154, 285, 289, 290), which contribute to the stabilization of different CalB substrate moieties.11 Structural changes to the three pockets upon adsorption to the triglyceride interface were quantified by monitoring the distance between the Cα atom of the catalytically active S105 and the Cα atoms of the amino acids constituting the acyl, medium, and large pockets (Figure 5). Distance deviations were analyzed for all CalB systems relative to the average structure from five independent 10 ns equilibration simulations in water starting from crystal structure 1TCA.12 Cα atom distance deviations remained within 0.5 nm in all systems, except for L144 in two simulations. Its adjacency to D145 suggests that L144 is part of a concerted motion with the hydrophilic amino acid of α5 which appears instrumental in

conformational change during an interval of 50 ns at t = 300 ns in the tricaproin (C6) system (Figure 3A). While simulating the detached state of CalB in aqueous solution, intermediate or closed conformations were predominant (Figure 4B). The active site entrance cleft was gradually closing from an open conformation for the CalB-C8-DET system, and was only partially closed for CalB-C4-DET and CalB-C6-DET (Figure 3B). The radius of gyration of Cα atoms of the helices (Figure S1) suggests that this could potentially be caused by local denaturation of helices α5 and α10 that occurs during adsorption onto the triglyceride−water interfaces. Local denaturation could also be partially responsible for the stronger fluctuations occurring in the detached systems. An additional potential factor could be stabilizing interactions between the protein and the interface that restrict conformational freedom in the adsorbed state. The shifts from open to closed states observed for the respective CalB systems confirm that the conformational space of α5 and α10 is accessible to the 1 μs time scale sampling by standard equilibrium MD simulations, revealing a bias toward open conformations when CalB is adsorbed to the triglyceride− water interface and to closed conformations when in aqueous environment (Figure 4). No striking systematic difference was thereby observed between the C4, C6, and C8 systems, although no definitive conclusions can be drawn from the present data due to a lack of replica simulations. Our interface models of atomistic resolution are thereby consistent with the previously reported CalB open13 and closed states15,24 and confirm the original assumption of helix α5 serving as a functional lid for CalB.12 The accessible conformational space of α5 and α10 suggests that the original CalB structure by Uppenberg et al.12 represents an intermediate conformational state under crystalline conditions. Recent crystal structures capturing open20 and closed24 conformations are more accurate depictions of the two environments characteristic of lipase and esterase activity, respectively. The molecular mechanisms that stabilize lid open and closed conformations in CalB have not yet been unequivocally established, and discussions range from single salt bridges12,24 to more generalized deliberations on the hydrophobic effect.27 Potential salt bridge formations were examined in the context of stabilizing CalB open and closed conformations. As α5 and α10 interact with the hydrophobic oil−water interface, they are D


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Figure 6. (A) During MD simulation, triglyceride−water interfaces self-assemble in aqueous solution not by orienting its polar moieties (dark gray) directly toward the aqueous environment (light blue) but by forming crystalline-like layers of segregated polar and nonpolar nanoenvironments in lamellar patterns. (B) The hydrophilic and hydrophobic lamellae are more pronounced and further spaced apart as the length of the acyl chain length increases: tributyrin (C4), tricaproin (C6), tricaprylin (C8). (C) CalB adsorbs in a productive orientation with its active site entrance facing the interface and thereby attaches with its predominantly hydrophobic helices α5 (green) and α10 (blue) to the nonpolar regions (light gray), and with the two hydrophilic amino acids (yellow circles) D145(α5) and K290(α10) binding to the polar region (dark gray).

Table 1. MAB Values Presented with Standard Deviations (STDs)a tributyrin (C4) helix α5

all Cα D145

helix α10

all Cα K290

MAB STD MAB STD MAB STD MAB STD

tricaproin (C6)

tricaprylin (C8)

hphil

hphob

hphil

Hphob

hphil

hphob

1.02 0.04 1.00 0.27 0.96 0.04 0.91 0.23

0.97 0.06 0.99 0.36 1.06 0.06 1.11 0.30

1.02 0.08 1.35 0.16 0.79 0.05 1.17 0.09

0.98 0.07 0.72 0.13 1.17 0.04 0.86 0.07

1.32 0.15 1.90 0.41 0.81 0.07 1.24 0.25

0.82 0.09 0.48 0.24 1.11 0.04 0.86 0.14

Moiety association bias (MAB)38 was calculated with a 1.1 nm cutoff for all Cα atoms of CalB helices α5 (residues 139−147) and extended α10 (residues 277−290) versus all polar atoms of the triglyceride ester group and the glycerol backbone (hphil), as well as versus all nonpolar atoms of the triglyceride acyl chains (hphob). The analysis was conducted on triglyceride−water interfaces composed of pure tributyrin (C4), tricaproin (C6), and tricaprylin (C8). MAB values are presented as averages over the final 250 ns of a 1000 ns simulation and presented with standard deviations (STDs). These helix-specific averages are compared to the hydrophilic amino acids D145(α5) and K290(α10).

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moieties and hydrophobic acyl chains were exposed to the aqueous environment. For tricaproin (C6) and tricaprylin (C8), the layer nanostructure resembled crystalline patterns of alternating lamellae consisting of aggregated polar and nonpolar moieties that extend perpendicularly from the interface into the bulk of the triglyceride (Figure 6B). This horizontal order of self-aggregated triglyceride molecules can be explained by a hierarchy of moiety association preferences. In water, triglyceride droplets form as a consequence of the preference of water to bind to other water molecules, thus maximizing hydrogen bonding.31,32 By the same principles, the triglyceride aggregate nanostructure forms as a consequence of the selfpreference of polar triglyceride moieties. The nanostructure of triglyceride assembly is thus a consequence of maximizing interactions between polar ester moieties within the aggregate nanostructure. To our knowledge, no experimental data exists on triglyceride nanostructure in aqueous emulsions that could corroborate these findings, since experimental methods to obtain data on nanostructures of condensed-phase emulsions are limited.33 However, data on solid state crystalline triglyceride aggregates show that triglyceride molecules stack in crystalline lamellar substructures34,35 that closely resemble

facilitating closed conformations. The adsorption of CalB appears to have a stabilizing effect on the polypeptide backbone of the active site (Figure S5). Adsorbed systems (Figure 5A) thereby resemble the crystal structure more closely than systems of CalB in aqueous solution (Figure 5B), which display significant discrepancies for the large pocket in particular, being indicative of closed conformations (Figures S6 and S7). The higher deviation of the large pocket is not surprising, since it is mostly formed by the mobile helices α5 (A141, L144) or α10 (I285, P289, K290). Thus, conformational changes of these helices not only change substrate accessibility but also result in changes of substrate stabilization in the large pocket. Triglyceride Self-Assembly Nanostructure Affects Conformations of Helices α5 and α10. The triglyceride− water interface models were created by extensive self-assembly simulations as described previously.30 Examining the nanostructure of the equilibrated triglyceride layers revealed a distinctive segregation of polar ester moieties and nonpolar aliphatic side chains (Figure 6A). In contrast to our expectations, the triglyceride molecules at the interface were not oriented with their polar groups toward the aqueous phase. Instead, clusters of hydrophilic ester E


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chain para-nitrophenyl (pNP) compounds is well established. Yagonita et al.42 found that V139E and A151D mutations yielded a 5-fold increase in activity against pNP caprylate. Circular permutations that significantly altered the substrate recognition or adsorption interface of CalB yielded multiple variants with increased activity toward pNP butyrate and DiFMU octanoate.43 Lid-grafting studies performed by Skjot et al. on pNP butyrate and laurate, as well as a diverse panel of esters, displayed significant changes in catalytic activity.15 Xie et al. deliberately targeted α10 for mutation and found that variation of L277, A281, and I285 resulted in large fluctuations in enzyme activity versus pNP caprylate;20 authors furthermore suggested that a balance between flexibility (α5 helix) and rigidity (α10 helix) in the active site is essential for catalysis. Amino acids peripheral to α5 and α10 on its flanking loops have also shown significant effects on CalB activity, with N292Y showing a 7.5-fold increase.44

the nanostructures that form in our simulations. Moreover, triglyceride emulsions consist of aggregate droplets ranging from several hundred to several thousand nm in diameter.36,37 Thus, the nanostructure of condensed-phase aggregates is expected to resemble the solid state. Previously, it was demonstrated that the observed nanostructure was also present in small triglyceride droplets of much higher curvature.38 The crystalline-like lamellar nanostructure of the triglyceride−water interface has significant implications on the conformations of adsorbed CalB. Since both helices α5 and α10 consist of predominantly hydrophobic amino acids, the helices are expected to preferentially interact with the nonpolar lamellae of the triglyceride nanostructure. Correspondingly, the two hydrophilic residues, D145(α5) and K290(α10), should preferentially associate with polar lamellae. This was supported by snapshots extracted from the simulation (Figure 6C) and by the moiety association bias MAB = pmeasured/prandom, calculated as the ratio between the measured probability pmeasured and the probability expected of a random molecular arrangement prandom.38 The moiety association bias statistically evaluates association preferences between different molecular moieties during simulation and was measured between residues of the helices α5 and α10 and polar and nonpolar moieties at the triglyceride surface (Table 1). While the MAB for all Cα atoms of α10 indicated a general preference toward nonpolar lamellae of tricaproin (C6) and tricaprylin (C8), both D145(α5) and K290(α10) prefer to associate with polar lamellae. Our findings corroborate the suggestions by Zisis et al. of a highly packed, self-assembled monolayer of uniform molecular arrangements as a sufficient condition for interfacial activation.13 We furthermore postulate a direct link between the nanostructure of the substrate selfassembly, the conformations of the helices forming the lid of CalB, and interfacial activation. During adsorption to the interface, polar and nonpolar amino acids of CalB interact with the respective polar and nonpolar lamellar nanostructures at the surface of the triglyceride self-assembly, which determine the position of α5 and α10. Since the spacing between segregated polar lamellae is determined by the length of the acyl moiety, the opening of the active site entrance channel depends on the substrate. Thus, interfacial activation can be considered as the result of an evolutionary adaptation of the protein lid structure to accommodate the nanostructure of the substrate interface in order to induce open conformations, facilitate substrate access, and optimize substrate stabilization near the active site. The dependence of lipase activity on the molecular structure of the interface was previously shown experimentally for heterofunctional supports with mixed hydrophilic and hydrophobic moieties.39,40 Another study on functionalized carbonbased nanomaterials found a correlation between enzyme activity and the degree of interface modification with hydrophilic moieties;41 the authors also observed subtle conformational variations and furthermore a more rigid structure for adsorbed lipase, whereas esterases displayed significant denaturation under the same conditions. These findings are in agreement with our observations. The specific recognition of a nanostructured substrate interface by the enzyme offers an interesting new enzyme engineering perspective for the design of lipases toward a particular substrate aggregate by optimizing specific open conformations that match the geometry of the nanostructured substrate. The influence of CalB mutations in helices α5 and α10 on esterase and lipase activity toward short-chain and long-

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CONCLUSION We have demonstrated open and closed conformations of the promiscuous enzyme Candida antarctica lipase B (CalB) in simulation models pertaining to lipase and esterase activity, respectively. The conformations were thereby identified by the relative motions of helices α5 and α10. The open conformation of adsorbed CalB was shown to be induced by the nanostructure of the triglyceride self-assembly, which displayed patterns of alternating polar and nonpolar lamellae. Binding of α5 and α10 to the nonpolar lamellae induces two major changes, an opening of the substrate access channel and a conformational change of the substrate binding site that results in a productive binding of a substrate molecule near the active site. In this regard, the nanostructured triglyceride acts as an allosteric effector of catalytic activity. We thus propose a link between open conformations, substrate stabilization, and the nanostructure of the triglyceride aggregate, which is optimized by evolution to fit the surface restraints of the natural substrate. This could explain the substrate dependency of interfacial activation of CalB. The highly mobile helices α5 and α10 of CalB are predominantly hydrophobic and contain only two polar amino acids, whereas the surface of the triglyceride−water interface displays a well-defined nanostructure with alternating polar and nonpolar lamellae. The anchoring of polar amino acids to the polar lamellae restricts the conformational space available to α5 and α10 and mediates the open conformation in the adsorbed state. The distance between α5 and α10, and thus the degree of cleft opening, directly correlates with the spatial gap between the polar lamellae of the self-aggregated triglyceride layer, which is a consequence of acyl chain length. Esterase activity of CalB is typical for aqueous environments, where α5 shifts toward α10, thus restricting both substrate access and substrate binding to small water-soluble substrates. However, α5 thereby maintains a high degree of flexibility, which may ensure substrate access regardless of closed conformations. The existence of CalB conformations that are specific to lipase (open) and esterase (closed) activity holds significant implications for the structure-based design of CalB, which relies on accurate representations of catalytically relevant structural conformations of the biocatalyst. The original CalB crystal structure,12 which is the most commonly referenced structure in CalB engineering studies, appears to represent an intermediate state between conformations of esterase and lipase activity and may thus not be adequate to assess mutations F


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the system and resolvating the same system with water, to reduce potential conformational biases by rehydration. Analysis of Distances and Conformations. To investigate the influence of CalB adsorption on triglyceride layers, the center of mass (COM) distance between helices α5 and α10 was monitored. Helix α5 was defined by residues 139−147, while the C-terminal of helix α10 (referred to as α10 in the following) was defined by residues 277−288. To define the α5 COM, only Cα atoms were taken into account (Figure 2A). Furthermore, the impact of protein adsorption on active site morphology was monitored by distances between the catalytic oxygen Oγ of S105 and Cα atoms of amino acid residues within 10 Å that protrude into the active site cavity. This incorporated all residues of the medium binding pocket for secondary alcohols (residues 39, 42, 47, 104, 225), the large binding pocket for secondary alcohols (residues 141, 144, 154, 285, 289, 290), as well as the acyl binding pocket (residues 134, 138, 157, 189, 190) (Figure 2B). Moreover, distances between residues D145−T158 and D145−K290 were monitored to investigate their recently reported role in stabilization of CalB open and closed conformations via hydrogen bonding and salt bridge formation (Figure 2C and D).24 Analysis of Conformations and Interfacial Nanostructure. To characterize the preferred conformations of helices α5 and α10 with respect to the nanostructure of the triglyceride layer, the moiety association bias was calculated.38 The moiety association bias (MAB = pmeasured/prandom) of moiety A toward moiety B was defined as the probability (pmeasured) of B being a nearest neighbor to A and is derived by counting atoms of moiety B within a sphere around moiety A, normalized by the total number of neighboring atoms observed during a simulation for all snapshots, divided by the probability expected for a random distribution (prandom = Nmoiety/Ntotal). The definition of random thereby assumes equal likelihood of any atom being a nearest neighbor to any other atom, and is equal to the number of atoms of a particular neighboring moiety (Nmoiety), divided by the total number of all atoms in the system (Ntotal).

for either lipase or esterase application. While structures of open20 and closed24 conformations were recently made available, a dedicated conformational survey on specific interfacial applications of CalB on a case-by-case basis would likely yield the best results.

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METHODS

Simulation Details. The GROMACS 5.0.4 software45 was used to model an NPT ensemble at 298.15 K and 1 bar. The Nosé−Hoover thermostat46,47 and the Parrinello−Rahman barostat48 were applied to simulate all systems for 1 μs, using the leapfrog integrator49 with a time step of 2 fs, constraining hydrogen bonds with LINCS.50 Periodic boundary conditions were applied in all three dimensions. Temperature coupling was isotropic and pressure coupling semi-isotropic, to decouple the x−y and z dimensions for improved accuracy in modeling the surface tension of the triglyceride layer.51 Long-range electrostatics were calculated with the particle mesh Ewald method.52 Lennard-Jones interactions were treated with a cutoff and capped at 1.2 nm. The OPLS-AA all-atom force field53 was used to parametrize CalB. Triglyceride molecules were parametrized with the Berger lipid model54 and adjusted in the 1−4 interactions with the half-ε doublepairlist method by Neale and Pomes to adjust for OPLS-AA force field parametrization. Water molecules were parametrized with the TIP3P55 model. The simulations were performed on the Hazelhen cluster, a Cray XC40 system of the High Performance Computing Center (HLRS) in Stuttgart, Germany. System Creation. The simulation systems of CalB adsorbed to triglyceride interfaces were prepared as outlined by Gruber et al.19 Thereby, systems were created that modeled helices α5 and α10 of CalB facing toward triglyceride layers within a distance of ∼6 Å. Thus, the adsorption process of CalB was modeled in its catalytically active conformation. Initially, CalB and the triglyceride layers were equilibrated separately. CalB (PDB: 1TCA)12 was modeled in a cubic aqueous system, maintaining all 286 crystal waters and neutralizing the net charge of −1 by adding a single Na+ ion. Equilibration was performed by two subsequent simulations: a 0.5 ns NPT simulation with restraints on CalB heavy atoms, starting from random velocities, and a subsequent 20 ns NPT simulation without restraints. Triglyceride layers (tributyrin, C4; tricaproin, C6; tricaprylin, C8) were created by randomly adding triglyceride molecules to a cubic system (Figure 1A) and then removing triglyceride molecules in one-half of a cubic system and solvating with water. After 500 ps NPT simulations with restraints on triglyceride atoms, the layer was thoroughly equilibrated for 300 ns without restraints to produce the layer, yielding a model interface of large-scale oil-in-water aggregates (Figure 1B). CalB−triglyceride−water systems were created by concatenating the cubic CalB−water and triglyceride−water systems. Before concatenation, the water volumes on the sides of the designated interface were reduced until approximately one water shell between the system boundary and the solute molecules remained. Thus, in the concatenated CalB−triglyceride−water systems, there were at least two water shells between CalB and the triglyceride layer. During concatenation, a small gap (≈2 Å) between the two boxes was introduced, to ensure that no water molecules overlap in the combined system. Thus, well-defined and comparable CalB−triglyceride−water systems were created with a distance of ∼6 Å between CalB and triglyceride. CalB was allowed to adsorb to the triglyceride layers in active orientation with distance restraints between Cα atoms of CalB to maintain the protein structure of the aqueous environment. 300 ns simulations were performed on these systems to ensure equilibration of CalB at the interface. The final configurations of these equilibration simulations were chosen as starting configurations for a 1 μs analysis simulation in the attached (ATT) state (Figure 1C). Additionally, after completing the analysis simulations for each of these three configurations, the protein structures were removed from the respective triglyceride layers and resolvated in water to create three starting configurations of simulations of the detached (DET) state (Figure 1D). This was done by deleting all triglyceride molecules from

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b04570. The radius of gyration of α5 and α10 from Candida antarctica lipase B (CalB) is supplied as Figure S1. An overview of CalB crystal structures and the distances referenced in the manuscript are supplied as Table S2 and Figure S3. A distance analysis of potential salt bridges stabilizing CalB open and closed conformations is supplied as Figure S4. Analysis of distance deviations between simulation data and crystal structure reference is supplied as Figure S5 for all CalB active site amino acids. A depiction of open and closed conformations found for CalB is supplied in Figures S6 and S7. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jürgen Pleiss: 0000-0003-1045-8202 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the German Research Foundation (DFG) for financial support of the project within the Cluster of Excellence in Simulation Technology (EXC 310/1) at the University of Stuttgart. We thank the high performance computing center G


Article

Langmuir

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Stuttgart (HLRS) for their support and for supplying the computational resources.

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