Article pubs.acs.org/biochemistry
Lipases That Activate at High Solvent Polarities Jakob Skjold-Jørgensen,†,‡ Jesper Vind,‡ Allan Svendsen,‡ and Morten J. Bjerrum*,† †
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark Novozymes A/S, Brudelysvej 35, DK-2880 Bagværd, Denmark
‡
S Supporting Information *
ABSTRACT: Thermomyces lanuginosus lipase (TlL) and related lipases become activated in low-polarity environments that exist at the water−lipid interface where a structural change of the “lid” region occurs. In this work, we have investigated the activation of TlL (Lipase_W89) and certain lid mutants, containing either a single positive charge mutation, E87K (Lipase_K87_W89), within the lid region or a lid residue composition of both lipase and esterase character (Hybrid_W89) as a function of solvent polarity. Activation differences between the variants and TlL were studied by a combination of biophysical and theoretical methods. To investigate the structural changes taking place in the lid region upon lipase activation, we used a fluorescence-based method measuring the efficiency of Trp89 in the lid to quench the fluorescence of a bimane molecule attached in front (C255) and behind (C61) the lid. These structural changes were compared to the enzymatic activity of each variant at the water− substrate interface and to theoretical calculations of the energies associated with lid opening as a function of the dielectric constant (ε) of the environment. Our results show that the lid in Lipase_K87_W89 undergoes a pronounced structural transition toward an open conformation around ε = 50, whereas only small changes are detected for Lipase_W89 ascribed to the stabilizing effect of the positive charge mutation on the open lid conformation. Interestingly, Hybrid_W89, with the same charge as Lipase_W89, shows a stabilization of the open lid even more pronounced at high solvent polarities than that of Lipase_K87_W89, allowing activation at ε < 80. This is further indicated by measurement of the lipase activity for each variant showing that Hybrid_W89 is more quickly activated at the water−lipid interface of a true, natural substrate. Combined, we show that a correlation exists between structural changes and enzymatic activities detected on one hand and theoretical calculations on lid opening energies on the other. These results highlight the key role that the lid plays in determining the polarity-dependent activation of lipases.
L
enzyme itself (enzyme theory).7−10 However, it is now the general consensus that both the substrate and the intrinsic conformational changes of the enzyme are important determinants for lipase activation. In TlL and related lipases, a small structural element called the “lid” (or flap) covers the active site, keeping the lipase inactive in a homogeneous aqueous solution. As the lipase encounters a water−lipid interface, the lid displaces itself from the active site triad, allowing substrate access to the active site and initiation of catalysis. The movement of the lid upon interfacial activation is difficult to investigate in vitro because the phenomenon takes place at the boundary between the water and lipid. Therefore, to gain an understanding of the structural dynamics involved in lipase activation, computer simulations have been widely used.11−15 These studies have supported the idea that the lid is closed when the lipase is present in a hydrophilic environment (high dielectric constant), whereas a structural transition to an open conformation occurs in a hydrophobic
ipases (EC 3.1.3) represent a special class of esterases that are capable of hydrolyzing ester bonds in water-insoluble triglyceride substrates. For many years, lipases have played an important role as industrial biocatalysts. Indeed, lipases from fungal sources such as Rhizomucur miehei, Thermomyces lanuginosus, Rhizopus delemar, Candida rugosa, and Candida antarctica A and B are today used extensively in applications such as detergents, foods, flavors, biodiesel production, and cosmetics.1,2 Specifically, Themomyces lanuginosus lipase (TlL) is a protein consisting of 269 amino acids and a member of the α/βhydrolase fold protein family3 with an active site triad comprising Ser146, His258, and Asp201.4 Characteristic for TlL and related lipases is the fact that their enzymatic activity is governed by a phenomenon termed “interfacial activation”. Interfacial activation, first described by Sarda and Desnuelle, refers to the inherent trait of lipases in which a large increase in activity is observed when the substrate reaches its critical micelle concentration (CMC) with very low activity toward the homogeneous, monomeric form of the substrate.5 This characteristic observation was earlier ascribed to either a change in the physical state of the substrate (substrate theory6) or a consequence of a conformational change within the © 2015 American Chemical Society
Received: October 13, 2015 Revised: December 1, 2015 Published: December 8, 2015 146
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environment (low dielectric constant).10,16,17 However, the specific solvent polarity at which lipase activation occurs has not previously been investigated experimentally. Advances within protein engineering have shown that the activation mechanism in TlL and related lipases can be altered by extensive mutagenesis of the residues constituting the lid region (residues 82−98, TlL numbering).18−23 Indeed, our previous studies have shown that it is possible to construct a TlL variant with a lid residue composition of both TlL and esterase (Hybrid) character that displayed faster activation at the water−lipid interface and showed activity toward watersoluble ester substrates higher than that of wild-type TlL.23 Recently, site-directed fluorescence labeling (SDFL) methods that allow the study of conformational changes taking place in proteins within 10−15 Å have been developed. Hence, these methods present certain advantages over Förster resonance energy transfer (FRET), which is generally limited to resolving structural changes above ∼20 Å,24 with a few exceptions.25−27 Indeed, the tryptopan-induced quenching (TrIQ) and tyrosineinduced quenching (TyrIQ) methods exploit the ability of aromatic residues [tryptophan (Trp) and tyrosine (Tyr), respectively] to quench the fluorescence of certain fluorophores attached to the protein body presumably through a photoinduced electron transfer (PET) process.28,29 In a recent study, we applied the TrIQ method to lipases showing that it is possible to probe differences in the degree of lipase activation (lid opening) between TlL and certain lid variants as a function of solvent polarity. In this study, we have expanded our investigation of the solvent-induced activation of TlL by combining both biophysical and theoretical methods. We have examined two lid variants, e.g., a variant containing a positive point mutation, E87K, within the TlL lid (Lipase_K87_W89) and a variant with a lid of both lipase and esterase character (Hybrid_W89), to elucidate the impact of charge and overall lid residue composition on TlL activation, respectively. Previous studies have shown that the E87K point mutation in the lid of TlL increases the level of stabilization of the open lid conformation in a hydrophobic environment,12 and Hybrid_W89 was chosen on the basis of previous results indicating that it becomes more quickly activated at the water−lipid interface than TlL.23,35 Importantly, TlL and the constructed variants contained a Trp residue in position 89 within the lid, making them suitable for TrIQ investigations. Thus, to investigate detailed, structural changes occurring in the lid upon activation, we labeled TlL and the lid variants with the Trp-sensitive fluorophore, bimane, behind (C61) and in front (C255) of the lid and measured bimane fluorescence quenching as a function of solvent polarity. To investigate the enzymatic activities of each mutant, we conducted activity assays on a semisoluble substrate, pNPbutyrate, below and above the critical micelle concentration (CMC) and pNP-decanoate embedded in a lipid layer of olive oil. Furthermore, MD simulations of lid opening were conducted according to previous methods10,12 measuring the stabilization of the open lid conformation as a function of the dielectric constant of the environment. We tested two hypotheses. (1) The E87K mutation in the lid of TlL allows lipase activation at high solvent polarities. Furthermore, a lid of both esterase and lipase character allows activation at even higher solvent polarities. (2) Activation studies in vitro correlate with in silico calculations.
Article
MATERIALS AND METHODS
Materials. Unless otherwise stated, all reagents and biochemical supplies were purchased from Sigma-Aldrich and affiliates. Variant Nomenclature. In this study, we named the constructed variants according to Table 1. Table 1. Features of the Constructed TlL Variants Used in This Study name
lid domain character
single-site lid mutations
quencher
Lipase_W89
TlL
none
Trp89
Lipase_K87_W89
TlL
E87K
Trp89
Hybrid_W89
none
Trp89
Esterase
both TlL and related hydrolases FAEA
none
Lipase_I89
lipase
W89I
Hybrid_I89
hybrid
W89I
no quencher no quencher no quencher
labeling site C61 or C255 C61 or C255 C61 or C255 C61 or C255 C61 or C255 C61 or C255
The character of the lid (lipase, hybrid, or esterase) and the quenching Trp (W) residue in position 89 are explicitly stated in the name of each variant. Hence, Lipase_K87_W89 refers to a variant containing a TlL lid, with a single E87K mutation and a Trp residue in position 89. Esterase, Lipase_I89, and Hybrid_I89 with no Trp in the lid were used as controls. All variants were made with a cysteine residue specifically incorporated into the backbone at modified sites G61C (behind the lid, C61) or I255C (in front of the lid, C255), allowing site-directed fluorescence labeling with the cysteine reactive fluorophore, monobromo bimane (mBBr). These labeling sites (C61 and C255) are not specified in the names of the mutants but are explicitly denoted in the figures presented herein. Variant Construction, Transformation, and Screening. Hybrid_W89 and Esterase variant genes were generated using splicing by overlap extension (SOE) polymerase chain reaction (PCR)30 as previously described.23 Lipase_K87_W89 and the single-cysteine site mutagenesis constructs were generated by site-directed mutagenesis according to the method of Weiner et al.31 Furthermore, Lipase_I89 and Hybrid_I89 were produced with a W89I mutation in the lid (see Figure S1 for the primary sequence alignment of the lid residues in each variant). Lipase variant genes were inserted into a cloning plasmid and transformed in competent Escherichia coli cells. Purified DNA was sequenced across the whole gene, transformed into an Aspergillus oryzae strain, and fermented according to previously described methods.32 Protoplasts were stored at −80 °C and thawed when needed. Expression was verified by running sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Variant screening was conducted for successfully transformed A. oryzae strains using a standard pNPvalerate activity assay.33 Purification. Expression and purification of lipase variants were conducted according to previous protocols23,34 using hydrophobic interaction and anion exchange chromatography methods. Expression was verified by SDS−PAGE using SimplyBlue Safestain (Life Technologies, catalog no. 147
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and spectral shifts were investigated for signs of ground state complex formation (and thus static quenching) between Trp (quencher) and the bimane fluorophore36 (Figure S2). Thermal Stability. The thermal stability assay was conducted as previously described.23,37 In brief, SYPRO Orange (SO) (catalog no. S5692, 5000× stock concentrate) was diluted 250 times in 50 mM MOPS (pH 7.5). Fifteen microliters of a dye solution was added to 15 μL of a 7 μM protein sample. Melting curves for each variant before and after labeling were determined using a StepOnePlus Real Time PCR System (Applied Biosystems) running a temperature gradient from 25 to 96 °C at a scan rate of 76 °C/h with an initial 15 min reaction time at 25 °C. Lipase Activity before and after Labeling. To elucidate the impact on labeling with bimane, the hydrolytic activity toward pNP-palmitate embedded in a lipid layer of rendered porcine fat (1:1 molar ratio, Dragsbæk A/S) was determined as previously described35 on labeled and unlabeled variants running the assay in 100 mM Tris (pH 8) and 2 mM CaCl2. Activity was calculated from the steepest increase in OD (at 405 nm) as a function of time. The assay was run at 22 °C. Interfacial Activation Assay. The interfacial activation of each variant was investigated running an activity assay using pNP-butyrate as the substrate as previously described.23,38 In brief, 190 μL of reaction buffer [a given concentration of pNPbutyrate in 25 mM MOPS (pH 7.5)] was added to a 10 μL protein sample (12 μM) in a nonbinding plate (Corning, catalog no. 3995) to minimize adsorption of the lipase to the well surface and hence lower artificial activity levels. The absorbance increase due to the released pNP moiety was monitored (Spectra Max Plus 384, Molecular Devices), and activity was calculated from the steepest increase in OD (at 405 nm) as a function of time at pNP-butyrate concentrations ranging from 0 to 6 mM. Lipase Assay. Lipase activity was determined by measuring the hydrolytic activity toward a pNP-decanoate embedded in a lipid layer of olive oil (1:1 molar ratio) as previously described.23 Activity was determined in 100 mM Tris (pH 8) and 2 mM CaCl2. The final protein concentration was 8 nM. The absorbance increase, due to the released pNP moiety, was monitored at 405 nm for 10 min. Calculation of the Dielectric Constant in Solvent Mixtures. The theoretical dielectric constants for the water/ ethanol solvent mixtures were calculated as previously described.35 In brief, using Oster’s rule,39 the polarization of a mixture of n components was determined from
LC6065). All samples were buffer-exchanged and concentrated in 50 mM MOPS (pH 7.5) using centrifugal filter units (Ultracel-10K, Millipore). Purification of Hybrid_I89 with labeling site C61 failed due to the low expression yield and high degree of binding to the column. Intact Molecular Weight Analysis. The intact molecular weight of each lipase variant was determined by LC−ESI-MS analysis using a MAXIS II electrospray mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) as previously described.35 In brief, all samples were diluted to ∼0.1 mg/mL in Milli-Q water. The intact molecular weights were determined for unlabeled and bimane-labeled samples. Samples were applied to an Aeris Widepore C4 column (Phenomenex), washed, and eluted running an acetonitrile linear gradient and introduced to the electrospray source with a flow of 300 mL/ min by an Ultimate 3000 LC system (Dionex). Data analysis was performed with DataAnalysis version 4.2 (Bruker Daltonik GmbH). Intact molecular weights of the unlabeled and labeled samples are shown in Table S1. Solvent-Exposed Hydrophobic Surface Area (SEHSA) Assay. The solvent-exposed hydrophobic surface area for each variant (unlabeled) was investigated using 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorophore, as previously described.35 In brief, the 0.2 mg/mL sample was mixed with 0.25 mM ANS in 25 mM MOPS (pH 7.5). The degree of ANS fluorescence was measured with excitation at 380 nm and emission collected from 400 to 650 nm. Excitation and emission slit widths were 10 nm. Measurements were recorded on a fluorescence spectrometer at room temperature (PerkinElmer, LS50B) in a quartz cuvette (Hellma QS 105.250). An average of three consecutive emission scans were taken for each measurement. All samples were reduced with ∼0.5 mM TCEP prior to analysis to remove any cysteinereacted species. Labeling of Lipase Variants. Variants were labeled with monobromo bimane (mBBR) according to a previous protocol.35 Briefly, 15 μM lipase [diluted in 25 mM MOPS (pH 7.6), buffer A] was treated with a 10-fold molar excess of TCEP to remove any cysteine-reacted species. The samples were incubated for 10−20 min at room temperature. mBBR was subsequently added in a 15-fold molar excess, and samples were incubated overnight at 4 °C or for 3−4 h at room temperature on a shaker bed in the dark. The reaction mix was subjected to gel filtration using NAP-5 (GE Healthcare) columns equilibrated with buffer A. Excess dye was removed using spin columns (10K molecular weight cutoff, AMICON ULTRA) washed with 5 column volumes of buffer A. This procedure has been shown to completely remove any unspecifically bound bimane from the labeled samples.35 The labeling efficiency was determined by measuring the absorbance spectrum of each variant (Shimadzu UV 1601) using an extinction coefficient for bimane of 5000 M−1 cm−1. The concentration of protein was calculated using extinction coefficients of 35560 M−1 cm−1 for Lipase_W89-based variants, 36840 M−1 cm−1 for Hybrid _W89-based variants, and 31150 M−1 cm−1 for Esterase-based variants and corrected for the change in the number of Trp residues using an extinction coefficient of 5690 M−1 cm−1 for a Trp residue. The labeling efficiency was between 20 and 114% (Table S3). Less than 5% unspecific labeling was observed using wild-type TlL (with no free, surface-exposed cysteine) as a negative control. UV/Vis. An absorbance spectrum from 250 to 700 nm (NanoDrop, Fisher Scientific) was measured for each variant,
n
pm =
∑i = 1 xivp ii n
∑i = 1 xivi
(1)
where pm is the polarization per unit volume and x, v, and p represent the mole fraction, molar volume, and polarization of pure component i, respectively. The dielectric constant was determined from the polarization per unit volume by40 p=
(ε − 1)(2ε + 1) 9ε
(2)
where p is the polarization per unit volume and ε is the dielectric constant of the fluid. From eqs 1 and 2, assuming that p equals pm, the dielectric constant for each solvent was determined. Note that these calculations are only approximations and assume no volume change upon mixing. The parameters and values for the calculated dielectric constants of 148
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Figure 1. (A) Helical wheels of the α-helix domain (residues 85−95, TlL numbering) in Lipase_W89, Lipase_K87_W89, Hybrid_W89, and Esterase showing the distribution of amino acid residues and the orientation of the amphipathic moments (black arrows) calculated on the basis of the hydrophobicity and side chain distribution of the amino acids in the helices.43 The helical wheel approximately represents the expected orientation of the α-helix domain when the lid is in its closed conformation (PDB entry 1DT3) covering the hydrophobic active site pocket (yellow arc). Accordingly, Lipase_W89, Lipase_K87_W89, and Hybrid_W89 show a distinct distribution of hydrophobic amino acids in the helix that generates a large amphipathic moment (long arrow) supposedly making favorable hydrophobic interactions with the hydrophobic active site. The αhelix domain sequence in Esterase contains fewer large, hydrophobic amino acids and displays a more randomized distribution of amino acids. This creates a low amphipathic moment (short arrow) and suggests that the lid in Esterase does not make hydrophobic interactions with the active site. The N- and T-termini of the helices are denoted with small red letters (N and T, respectively). The location of Trp89 in the helices is indicated with red letters. The calculations and representations of the α-helical properties were made using HeliQuest software tool43 [see the Supporting Information for calculations of the hydrophobicities and amphipathic moments of each α-helix (Table S2)]. (B) View of the α-helix domain (residues 85−95) in the inactive (closed lid) crystal structure of TlL (PDB entry 1DT3) covering the active site pocket (translucent yellow surface) and catalytic triad (pink sticks) with residues shown as colored sticks. Residue legend: red for Asp (D) and Glu (E), purple for Thr (T) and Ser (S), blue for Lys (K), pink for Asn (N) and Gln (Q), yellow for Tyr (Y), Trp (W), Phe (F), Iso (I), and Leu (L), and gray for Ala (A) and Gly (G). The boundary between the hydrophobic and hydrophilic faces of the helix is shown with a dashed line. No distinct boundary is apparent for Esterase. The residue in position 95 is not shown because it is not part of the α-helix domain in the crystal structure.
constant of the environment. In brief, crystal structures of TlL with a closed and open lid (PDB entries 1DT3 and 1EIN, respectively42) were used as models for the inactive and active structures, respectively. All atoms within the lid region (residues 82−98) and atoms within 6 Å of any residue in the lid region were free to move. The conformational change of the lid going from a closed to an open state was simulated by restraining pseudotorsional dihedral angles (torsion angles between planes of four consecutive Cα atoms) of the lid region in 20 steps between the closed and open conformations of the lid.42 Prior to each simulation, the structures were optimized by applying 200 steps of steepest descent energy minimization and 600 steps of conjugate gradient energy minimization. Simulations were started using a time step of 1 fs and a simulation time of 5 ps. The mean energy differences between the open and closed conformations (step 20 and step 1) were calculated at each dielectric constant [using a constant dielectric constant (CDIE)] from ε = 4 to ε = 80.
the applied water/ethanol solvent mixtures are listed in Table S4. Steady State Fluorescence Measurements. The fluorescence emission and excitation measurements were taken on a fluorescence spectrometer at room temperature (PerkinElmer, LS50B) in a quartz cuvette (Hellma QS 105.250) at 22 °C. Excitation and emission slit widths were set to 10 nm. Experiments were conducted at 22 °C. Emission spectra were measured from 400 to 700 nm with excitation at 390 nm. Integrated steady state fluorescence emission intensities were calculated by integrating the fluorescence spectra from 410 to 700 nm. Structural Modeling of Bimane-Labeled TlL. Crystal structures of the inactive (lid closed) and active (lid open) TlL [Protein Data Bank (PDB) entries 1EIN and 1DT3, respectively] were used to visualize the bimane covalently attached to C61 and C255 on the protein body using Discovery Studio Visualizer.41 To elucidate the preferred orientation of the bimane fluorophore at each labeling site in the open and closed form of TlL, structures were energy minimized by using the Smart Minimizer algorithm and applying the CHARMm force field in Discovery Studio. MD Simulation. The constraint molecular dynamic simulation protocol previously described10,12 was used to investigate the energy landscape of lid opening (lipase activation) in each of the variants as a function of the dielectric
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RESULTS The Amino Acid Content and Distribution in the Lid of TlL and Lid Variants Reveal Differences in α-Helical Hydrophobicity and Amphiphilicity. The α-helical domain of the lid region in TlL and lid variants was investigated using HeliQuest (Figure 1).43 For this analysis, the α-helix domain was defined by residues 85−95 (TlL numbering). 149
DOI: 10.1021/acs.biochem.5b01114 Biochemistry 2016, 55, 146−156
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Biochemistry The residues comprising the α-helix domain in Lipase_W89, Lipase_K87_W89, and Hybrid_W89 create a large amphipathic moment perpendicular to the α-helix axis due to the distinct distribution of hydrophobic residues in the helix. In contrast, Esterase (with a FAEA-like lid) displays a very low amphipathic moment (μH = 0.108) caused by a random distribution of hydrophobic and hydrophilic amino acids in the α-helix. The orientation of the helical wheels corresponds well to the orientation of the lid in the crystal structure of inactive (closed lid) TlL [PDB entry 1DT3 (Figure 1B)] covering the active site. Because the amphipathic moment of a helix represents its tendency to undergo a preferred orientation between a polar and a nonpolar medium,44 these results suggest that the α-helix in Lipase_W89, Lipase_K87_W89, and Hybrid_W89 favors a closed state in polar solution where the lid can make favorable hydrophobic interactions with the hydrophobic, active site pocket.4 Conversely, little preferred orientation of the helix in Esterase would be expected given its low amphipathic moment, suggesting that Esterase is more likely to have an open lid conformation in a homogeneous aqueous solution. Labeling with Bimane Does Not Perturb Enzymatic Activity or Thermal Stability. We investigated the impact of bimane labeling by conducting thermal stability and hydrolytic activity assays on all samples. The assays did not indicate any severe perturbations in stability or activity after labeling (Figures S3 and S4). It should be noted that the labeling efficiency was quite low for some of the labeled variants (around 0.2), and reported activities and stabilities thus represent contributions from both nonlabeled and labeled enzymes (see Table S3 for labeling efficiencies). The Lid Remains Closed for Lipase_W89, Lipase_K87_W89, and Hybrid_W89 in Homogeneous Aqueous Solution As Suggested by the Solvent-Exposed Hydrophobic Surface Area. The surface-exposed hydrophobic surface area (SEHSA) for each lipase variant (unlabeled) was determined using 1,8-ANS as an extrinsic fluorophore. As our previous studies have indicated, Esterase [with a lid region of ferulic acid esterase (FAEA) character] has an open lid conformation in homogeneous aqueous buffer exposing the hydrophobic active site pocket. Hence, Esterase was used as a reference for the ANS fluorescence emission intensity measurements. As seen from Figure 2, the fluorescence emission intensity of ANS for Lipase_W89 was negligible relative to that of Esterase, indicative of an absence of exposed hydrophobic patches or ion pairing groups on the protein surface. The magnitude of the ANS signal for Lipase_K87_W89 was also relatively low (15%) albeit higher than that of Lipase_W89 ascribed to the E87K mutation that, in principle, could lead to ion pair formation with the sulfonate group in the naphthalene system of ANS.45 Interestingly, Hybrid_W89 displayed a significantly stronger signal (40 and 20% compared to Esterase for C61 and C255 mutants, respectively). Given that there is no additional positive charge mutation in Hybrid_W89 compared to Lipase_W89, the increased magnitude of the ANS signal is ascribed to a reduced level of gating of the active site by the lid domain, exposing the hydrophobic active site patch in homogeneous aqueous solution. Lipase_K87_W89 and Hybrid_W89 Show Higher Activities on pNP-Butyrate below and around the Critical Micelle Concentration Compared to Lipase_W89. TlL and most other lipases become activated when
Figure 2. Variants display different amounts of surface-exposed hydrophobic surface area (SEHSA) as indicated by differential binding of the extrinsic fluorophore, 1,8-ANS. (A) Structural models of TlL in its inactive (closed lid) and active (open lid) conformations (PDB entries 1DT3 and 1EIN, respectively;42 black α-helices, surface representation; oxygen, red; nitrogen, blue; carbon, green; residues 146−152, 172−179, 201−217, and 251−258) showing the hydrophobic surface area exposed upon lid opening (activation). (B) Fluorescence emission spectra of ANS bound to TlL variants with a cysteine at site C61 (left) or C255 (right) (unlabeled). The emission spectra were normalized using Esterase as a reference (green curve). Spectra (black curve) represent an average of three consecutive measurements. The experiment was conducted at room temperature in 25 mM MOPS (pH 7.5). The spectra from C255 variants were taken from ref 35.
the substrate reaches its critical micelle concentration (CMC) forming a water−lipid interface within which the lipase reaches its full catalytic potential. To probe the interfacial activation in each mutant, activity was determined as a function of pNPbutyrate concentration according to a previous protocol23,38 (see Figure S5 for activity plots). Lipase_W89, Lipase_K87_W89, and Hybrid_W89 all displayed interfacial activation as the concentration of pNP-butyrate exceeded its CMC and displayed low activity below this point. Hence, these results suggest that the mutants, like TlL, are “true” lipases, preferably acting on water-insoluble substrates. However, as seen from Figure 3, the relative activity levels determined just around the point of CMC reveal that Lipase_K87_W89 and Hybrid_W89 have activities significantly higher than that of Lipase_W89 between 0.8 and 2 mM pNP-butyrate. For C255 and C61 mutants, the activities of Lipase_K87_W89 and Hybrid_W89 were between 25 and 30% and between 25 and 35%, respectively, at 2 mM pNP-butyrate relative to the activity level measured at 6 mM pNP-butyrate. In comparison, Lipase_W89 and wild-type TlL displayed only between 0 and 5% of full activity at this substrate concentration. These results suggest that a higher fraction of open conformers are 150
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Figure 3. Activity of each variant as a function of pNP-butyrate concentration for (A) C61 (unlabeled) and (B) C255 variants (unlabeled) relative to the activity measured at 6 mM: Lipase_W89 (dark gray), Hybrid_W89 (red), and Lipase_K87_W89 (blue). Also included are the wild-type TlL activities (no mutations made, light gray). As shown, Lipase_K87_W89 and Hybrid_W89 display activities below and around the CMC of pNP-butyrate higher than that of Lipase_W89 and TlL (wild-type). Error bars denote the standard deviation from duplicate measurements.
Figure 4. Structural models of inactive and active TlL with the lid (dark gray cartoon) in the closed and open conformation (gray ribbon, PDB entries 1DT3 and 1EIN, respectively) and with bimane attached to sites C61 (A and B) or C255 (C and D) on the protein backbone. (A) In the closed state, Trp in position 89 is pointing toward the active site pocket, allowing intimate interaction with bimane at C61 (strong interaction). (B) In the open state, Trp is exposed to the solvent and the α-helix blocks interaction with bimane at C61 (weak interaction). (C) In the closed state, Trp is “out of sight” of bimane at C255 (weak interaction). (D) In the open state, Trp confronts bimane at C255 (strong interaction). The bimane’s empirically determined “sphere of quenching”51 [representing the Cα−Cα between the bimane labeling site (blue sphere) and the quencher (W89)] within which static [pink sphere; radius (r) = 8 Å] and dynamic quenching [gray sphere; radius (r) = 10−15 Å] can occur, respectively, is shown for each labeling site. Residues of the active site (AS) triad are shown as pink sticks.
transiently present below the critical micelle concentration of pNP-butyrate for Lipase_K87_W89 and Hybrid_W89. Structural Models. We chose to investigate the structural changes occurring in the lid domain upon lowering the solvent polarity by employing the TrIQ method and measuring the efficiency of Trp89 (within the lid of each mutant) to quench the fluorescence of bimane attached in front (site 255) or behind (site 61) the lid, respectively. We have previously shown that lid opening can be probed between bimane attached to site 255 on the protein backbone and Trp89 within the lid.35 In this study, site 61 was chosen in addition to site 255 to probe lipase activation from both sides of the lid, allowing a more reliable detection of structural changes. Accordingly, two sets of variants were made: (1) variants containing a labeling site behind the lid (C61) and (2) variants containing a labeling site in front of the lid (C255) (Figure 4). To visualize the preferable orientation of bimane when it is covalently linked to these sites, energy minimization was conducted on bimane-labeled structures of inactive (closed lid) and active (open lid) TlL (PDB entries 1DT3 and 1EIN, respectively). As seen from Figure 4A, bimane attached to C61 points toward the active site and interacts intimately with Trp89 when the lid is closed. In contrast, in this lid conformation, bimane attached to site C255 is “out of sight” of Trp89 (Figure 4C). As the lid opens, the Trp89 moves away from bimane at C61 (Figure 4B) and ultimately confronts bimane at C255 (Figure 4D). Altogether, these structural models were used to predict the bimane−Trp interaction pattern going from the inactive (closed lid) to the activated (open lid) state in TlL. The Lid Moves toward an Open State When the Solvent Polarity Is Lowered As Revealed by Tryptophan-Induced Quenching in Water/Ethanol Mixtures. The conformational state of the lid as a function of the dielectric constant (ε) of the solvent was measured using the TrIQ method as previously described. 35 The bimane fluorescence intensities of each variant (with bimane at C61 or C255) were measured in water/ethanol solvent mixtures of
theoretically determined dielectric constants between ε = 46 and ε = 80 (Figure 5A). For variants with a labeling site at C61 (behind the lid), the bimane fluorescence for Lipase_W89 and Lipase_K87_W89 was substantially quenched in buffer alone (60 and 75%, respectively), suggesting an intimate interaction between bimane and Trp89 in the lid indicative of a closed lid conformation in homogeneous aqueous buffer [ε = 80 (Figure 5B, I)]. Interestingly, Hybrid_W89 displayed a significantly higher fluorescence emission intensity indicative of a weakened interaction between bimane and Trp89 (40% quenching) in aqueous buffer (Figure 5A). Large structural movements of the lid toward an open conformation (Figure 5B, II) were observed for Lipase_K87_W89 between ε = 80 and ε = 46, whereas Lipase_W89 showed much less movement at these solvent polarities. Indeed, the fluorescence intensity nearly reached unity at ε ∼ 46 for Lipase_K87_W89, whereas the bimane intensity for Lipase_W89 changed to only a small extent (55% fluorescence intensity) at this solvent polarity. Interestingly, with only 40% bimane quenching, Hybrid_W89 showed less interaction with bimane attached to site C61 in buffer alone [ε = 80 (Figure 5A)]. Hence, this indicates that Trp89, on average, is less in contact with the bimane probe, which would suggest an increased tendency of the lid to move toward an open conformation (Figure 5B, II). Also, as the solvent polarity was decreased, the bimane fluorescence intensity increased to unity in Hybrid_W89, similar to that in Lipase_K87_W89. For lid variants labeled at site C255, bimane quenching was nearly the same (∼40%) for Lipase_W89, Lipase_W89_K87, 151
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Figure 5. (A) Bimane fluorescence emission intensities of bimane labeled at site C61 (red line) and C255 (black line) as a function of dielectric constant of the solvent for Lipase_W89, Lipase_K87_W89, and Hybrid_W89. The intensities (F) were normalized to the bimane fluorescence (F0) intensities measured for the Trp-less control (Lipase_I89) in each water/ethanol mixture. For C61 mutants, bimane fluorescence quenching decreased as the solvent polarity decreased. For C255, the fluorescence quenching increased with solvent polarity in accordance with previous studies.35 Experiments were conducted at room temperature. Error bars denote the standard deviation from two separate experiments. The data points of the black line are representative of a single experiment conducted in triplicate. For comparison, we have included the bimane fluorescence quenching data from previous studies35 for Lipase_W89 labeled at site C255 showing error bars of the standard deviation from two separate experiments (blue line). Corresponding steady state fluorescence emission spectra are given in Figure S6. Numbers within the plots refer to the (B) structural models of the different scenarios of the Trp−bimane interaction in the closed and open TlL (dark gray cartoon, PDB entries 1DT3 and 1EIN, respectively): (I) closed lipase labeled at C61, (II) open lipase labeled at C61, (III) closed lipase labeled at C255, and (IV) open lipase labeled at C255. Structures were visualized and energy minimized using the Discovery Studio Visualizer software suite.41 Cα−Cα distances between Trp and bimane are shown as stippled red lines.
difference (Enth step − Eclosed) for each opening step (n). Figure 6B shows an example of an energy difference plot calculated for a lid opening simulation at ε = 58 for the four variants. As the lid opens, a maximal energy difference is reached (around steps 10−12), corresponding to a lid conformation that is halfway open. At subsequent steps, the energy difference decreases, which is indicative of a more stabilized lid conformation. The energy difference between the fully closed (inactive) and fully open (active) conformations for each variant was calculated at 19 dielectric constants ranging from 4 to 80 (see Figure 6C). ε = 4 represents the polarity in a highly apolar environment, whereas ε = 80 represents the approximate dielectric constant of pure water.46 For Lipase_W89, the open lid conformation (negative energy difference) was stabilized around ε ∼ 10 similar to Lipase_K87_W89, which showed an open lid conformation stabilized at ε < 20. However, Lipase_K87_W89 displayed a general decrease in the calculated energy differences compared to Lipase_W89 throughout the entire range of dielectric constants. These results support previous findings signifying the importance of electrostatic interactions in stabilizing the open lid conformation.12 Interestingly, Hybrid_W89, with no additional charges in the lid region compared to Lipase_W89, showed an even greater stabilization of the open lid conformation at all dielectric constants ascribed to its partial esterase lid character. This was supported by the energy differences calculated for Esterase (containing a full esterase-like lid) showing that the open lid conformation is strongly preferred throughout the dielectric constant range. Our results are in line with the presented fluorescence quenching measurements suggesting
and Hybrid_W89 in buffer alone, indicative of a fairly weak Trp−bimane interaction (Figure 5A). As the dielectric constant of the solvent was lowered, the quenching increased for all variants, indicating that Trp89 moves toward the bimane probe in accordance with the proposed structural model (Figure 5B, IV) and our previous findings.35 Hence, the quenching data from bimane labeled at sites 61 and 255 cooperatively suggest that the lid moves toward an open, activated conformation when the dielectric constant of the solvent is lowered as demonstrated by the bimane fluorescence quenching patterns. Together, these results suggest that Lipase_K87_W89 and Hybrid_W89 become activated at solvent polarities higher than that of Lipase_W89. As is evident from Figure 5A, it seems that labeling site C61 is best suited for detecting structural changes occurring in the lid as a function of solvent polarity. As our previous studies have shown, only minute differences in activation could be detected using C255 as a labeling site. Hence, we recommend site 61 as the bimane labeling site for future investigations of structural changes taking place in the lid of TlL. MD Simulations Indicate That Hybrid and Lipase_K87_W89 Activate More Favorably in Polar Solutions Than Lipase_W89. MD simulations were conducted to investigate the energy profile of lid opening for each variant as previously described.12 In short, 20 pseudotorsional dihedral angles were defined between the closed and open conformations of the lid in TlL (PDB entries 1DT3 and 1EIN, respectively), and thus, lid opening was achieved by forcing the lid through these defined angles (steps) (Figure 6A). Starting in the closed lid conformation (inactive), we calculated the energy 152
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Figure 6. MD simulations on lid opening in TlL and constructed lid variants. (A) Structural model used in the lid opening simulations. Lid opening was investigated by forcing the lid to open through 20 defined pseudotorsional dihedral angles (steps). Residues in the active site are shown as pink sticks. (B) Plot of the energy difference (Enth step − Eclosed) at each opening step (n) at ε = 58 for each variant: Lipase_W89 (dark gray squares), Lipase_K87_W89 (blue diamonds), Hybrid_W89 (red triangles), and Esterase (white dots). (C) Total energy difference (Eopen − Eclosed) at 19 dielectric constants ranging from 4 to 80 calculated from the energy difference between step 20 and step 1: Lipase_W89 (dark gray dots), Lipase_K87_W89 (blue dots), Hybrid_W89 (red dots), and Esterase (white dots). A negative energy difference suggests stabilization of the lid conformation at the specified dielectric constant. Error bars denote the standard deviation from three separate simulations. For total energy plots of C61 and C255 mutants, see Figure S7.
that Hybrid_W89 and Lipase_K87_W89 can undergo an energetically favorable structural transition toward an open lid conformation at solvent polarities higher than that of Lipase_W89. Activation at the Water−Lipid Interface Is Faster for Hybrid_W89 Than for Lipase_W89 and Lipase_K87_W89. To investigate activation in the presence of a strictly water-insoluble, natural lipase substrate, we measured the enzymatic activity toward pNP-decanoate embedded in a layer of olive oil as previously described23 (Figure 7). Interestingly, TlL (wild-type, no labeling site), Lipase_W89, and Lipase_K87_W89 displayed a characteristic lag phase47 (∼3 min) before reaching their full catalytic potential, whereas Hybrid_W89 showed immediate catalysis at the water−lipid interface for both the C61 and C255 mutants. These results are in accordance with previous studies of the activation mechanism of the Hybrid variant23 suggesting that Hybrid is more quickly activated at the water−lipid interface of a natural substrate than Lipase_W89 and Lipase_K87_W89.
Figure 7. Normalized lipase activity plotted as the increase in the optical density (OD) of hydrolyzed pNP-decanoate embedded in a lipid layer of olive oil as a function of time for (A) C61 and (B) C255 variants (unlabeled): Lipase_W89 (dark gray squares), Hybrid_W89 (red circles), Lipase_K87_W89 (blue triangles), and wild-type TlL (light gray diamonds). Hybrid_W89 seems to activate immediately at the water−lipid interface, whereas wild-type TlL, Lipase_W89, and Lipase_K87_W89 display a lag phase of several minutes (∼3 min) before reaching their highest catalytic rate. The assay was conducted in 100 mM Tris (pH 8) and 2 mM CaCl2 at 22 °C. Error bars denote the standard deviation from triplicate measurements.
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DISCUSSION In this study, we have investigated lipase activation using a combination of methods measuring enzymatic activity, detecting structural changes using the tryptophan-induced quenching method and calculating the lid opening energies using an MD simulation protocol. On the basis of our results, we report that both Lipase_K87_W89 and Hybrid_W89 can undergo a structural transition toward an open state that is less
energy demanding than Lipase_W89 at high solvent polarities. Our results show that a correlation exists between activation studied in vitro and in silico as a function of the dielectric constant (polarity) of the solvent. Accordingly, of the lipases 153
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Biochemistry studied, activation occurs from high to low solvent polarity in the following order: Hybrid_W89, Lipase_K87_W89, Lipase_W89. Peters et al. have previously shown that Glu87 in TlL serves to stabilize the lid in a closed conformation (in polar solution) because of unfavorable electrostatic interactions with Asp62 in the open conformation. Mutating this residue to a lysine increased the energy gain upon activation favoring an open lid at low dielectric constants.12 Our studies support these findings and suggest that the stabilizing effect of the E87K mutation, although diminished in magnitude, is pronounced in highpolarity environments, as well. Accordingly, we have shown that the lid in Lipase_K87_W89 undergoes structural transitions toward an open state at ε ∼ 50. Hybrid_W89 displayed an even greater stabilization of the open lid conformation at dielectric constants. Of the 17 residues that comprise the lid region, Hybrid_W89 contains eight site specific residues that differ from those of TlL. Besides an E87T mutation (removing a negative charge that disfavors an open lid conformation in polar solution), Hybrid_W89 also contains mutations, G91L and N92D, in the α-helix domain of the lid that are similar to those found in Esterase. In addition to affecting the energies associated with lid opening (electrostatic, van der Waals, hydrogen bonding, etc.), these mutations lower the amphipathic moment of the α-helix domain, which suggests that the lid has a weaker tendency to orient itself in a preferred orientation between a polar medium and an apolar medium. Indeed, as suggested from the increased degree of ANS binding and higher activity levels below the CMC of pNP-butyrate, an increased fraction of open conformers of Hybrid_W89 are transiently present in a homogeneous aqueous solution compared to those of Lipase_W89 and Lipase_K87_W89. Furthermore, Hybrid_W89 contains a proline mutation in the anterior hinge domain (H2) of the lid region. As suggested by Shu et al., prolines in the hinge domain of Aspergillus niger serve to stabilize the open lid conformation.48 Hence, it seems that both the α-helix domain and the anterior hinge domain of Hybrid_W89 play important roles in stabilizing the lid at high solvent polarities. In the lipase assay, Lipase_W89 and Lipase_K87_W89 showed a distinct lag phase (induction time) before reaching their maximal catalytic rate. Hence, despite the stabilizing effect of the E87K mutation, these results suggest that it does not leverage faster activation at the water−lipid interface of a true, lipase substrate. Conversely, immediate activity was observed for Hybrid_W89. We ascribe these findings to the increased stability of the open lid conformation at high solvent polarities. Hence, the split personality of Hybrid_W89, with both lipase and esterase features, indicatively facilitates faster activation ascribed to the energies associated with lid opening, the amphipathic moment of the α-helix, and the proline mutation in the H2 domain.
Hybrid_W89 containing multiple mutations in the lid displays faster activation at the water−lipid interface of a triglyceride substrate ascribed to an increased level of stabilization of the open lid conformation at high solvent polarities.
CONCLUSION We have investigated the activation mechanism in TlL using a combination of methods to elucidate enzymatic activity, structural changes, and energies of lid opening at different solvent polarities. Our results support previous work underlining the lid’s essential role in governing enzymatic activity and interfacial activation.19,49,50 Furthermore, our findings show a correlation between in vitro and in silico experiments suggesting that mutagenesis of the lid in TlL can lower the energy barrier associated with lid opening. Specifically, we have shown that
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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.biochem.5b01114. A figure showing the primary sequence alignment of the lid regions of the lipases investigated in this study, a table showing the determined molecular weights of unlabeled and bimane-labeled lipase variants, a figure showing the absorbance spectra of C61B mutants, a table showing the calculated hydrophobicity, amphipathic moment, and charge of the α-helix in the lid region of each variant, a table showing the labeling efficiency of each bimanelabeled lipase, a figure of the melting temperatures for each variant before and after labeling, a figure showing the specific enzymatic activities of each variant before and after bimane labeling, a figure showing the activity of each variant toward pNP-butyrate as a function of substrate concentration, a figure showing the fluorescence emission spectra of each variant labeled with bimane at C61 or C255 as a function of solvent polarity, a figure showing the MD simulation plots of the total energy differences in lid opening for each variant, and a table showing the numbers used for calculating the dielectric constant of water/ethanol solvent mixtures (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Funding
This work was funded by the industrial Ph.D. program EFU:Novozymes (12-128740 and 0604-01457B). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the scientists at Novozymes, R&D. Special thanks to Christian I. Jørgensen, Clive Owens, and Peter R. Eriksen for technical guidance in mass spectrometry. Thanks to Esben Friis for assistance with computer-related issues.
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ABBREVIATIONS CMC, critical micelle concentration; MD, molecular dynamics; TrIQ, tryptophan-induced quenching; pNP, p-nitrophenol; TlL, T. lanuginosus lipase; FAEA, ferulic acid esterase from A. niger; IA, interfacial activation.
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REFERENCES
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