Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions

It is generally expected that 2-pore domain K⁺ (K_2P) channels are open or outward rectifiers in asymmetric physiological K⁺ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K_2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K_2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K_2P channels display 2 distinct types of I-V curves in asymmetric physiological K⁺ gradients. Two K_2P isoforms in the TWIK subfamily conduct large inward K⁺ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K_2P subfamilies conduct small inward K⁺ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K_2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K_2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K_2P channels and suggest that TWIK K⁺ channels have a unique feature in regulating cellular function.     

Selectivity of cytosolic phospholipase A2 type IV toward arachidonyl phospholipids

Cytosolic phospholipase A2 (cPLA₂) is an interesting protein involved in inflammatory processes and various diseases. Its catalytic mechanism as well as its substrate specificity for arachidonyl phospholipids is not typical for other phospolipases. Furthermore, a lid structure, which ensures a hydrophilic surface of the protein without any substrate bound and the movement of this flexible loop to make the hydrophobic active site accessible, is of high interest. Therefore, the focus of this work was to determine the binding mode of cPLA₂ with various substrates, such as arachidonic acid, a synthetic inhibitor, a saturated phospholipid, and most importantly an arachidonyl phospholipid. To understand the selectivity of the protein toward the arachidonyl phospholipid and the interaction in a protein–ligand complex, molecular dynamics simulations were performed using the GROMOS suite of simulation programs. The simulations provide insight into the protein and showed that selective binding of arachidonyl phospholipids is because of the shape of the sn‐2 tail. The amino acids Asn555 and Ala578 are involved in the strongest interactions observed in the protein–ligand complexes. Copyright © 2015 John Wiley & Sons, Ltd.     

A robust all-atom model for LCAT generated by homology modeling

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A₂ (PLA₂) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA₂. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.     

Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.     

Engineering of Yarrowia lipolytica lipase Lip8p by circular permutation to alter substrate and temperature characteristics

Applications of lipases are mainly based on their catalytic efficiency and substrate specificity. In this study, circular permutation (CP), an unconventional protein engineering technique, was employed to acquire active mutants of Yarrowia lipolytica lipase Lip8p. A total of 21 mutant lipases exhibited significant shifts in substrate specificity. Cp128, the most active enzyme mutant, showed higher catalytic activity (14.5-fold) and higher affinity (4.6-fold) (decreased K _m) to p-nitrophenyl-myristate (pNP-C₁₄) than wild type (WT). Based on the three-dimensional (3D) structure model of the Lip8p, we found that most of the functional mutation occurred in the surface-exposed loop region in close proximity to the lid domain (S112–F122), which implies the steric effect of the lid on lipase activity and substrate specificity. The temperature properties of Cp128 were also investigated. In contrast to the optimal temperature of 45 °C for the WT enzyme, Cp128 exhibited the maximal activity at 37 °C. But it is noteworthy that there is no change in thermostability.     

Phylogenetic analysis and in-depth characterization of functionally and structurally diverse CE5 cutinases

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications, however, requires deeper knowledge of cutinases’ biodiversity and structure–function relationships. Here, we mined over 3000 members from carbohydrate esterase family 5 for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis, which showed that cutinases with available crystal structures were phylogenetically closely related. We then selected nine phylogenic diverse cutinases for recombinant production and characterized their kinetic activity against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C₂ to C₁₆). Each investigated cutinase had a unique activity fingerprint against the tested pNP substrates. The five enzymes with the highest activity on pNP-C₁₂ and C₁₆, indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure–function analysis. All five enzymes showed a decrease in k_cat values with increasing substrate chain length, whereas K_M values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K_M values, resulting in high catalytic efficiencies toward pNP-C₁₆. Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.     

An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity

Benzoic acid esterases and ferulic acid esterases (FAE) are enzymes with different profiles of substrate specificity. An extracellular esterase (EstBC) from culture supernatants of the edible basidiomycete fungus Auricularia auricula-judae was purified by anion exchange chromatography, followed by preparative isoelectric focusing and hydrophobic interaction chromatography. EstBC showed a molecular mass of 36 kDa and an isoelectric point of 3.2 along with broad pH and temperature windows similar to fungal FAEs. However, EstBC exhibited also characteristics of a benzoic acid esterase acting on both benzoates and cinnamates, and most efficiently on methyl and ethyl benzoate, methyl 3-hydroxybenzoate and methyl salicylate. Feruloyl saccharides as well as lipase substrates, such as long chain fatty acids esterified with glycerol, polyethoxylated sorbitan and p-nitrophenol were not hydrolyzed. Protein database analyses with tryptic peptides of EstBC solely yielded hits regarding hypothetical proteins belonging to the alpha/beta hydrolase family. The uncommon substrate specificity of EstBC concomitant with a lack of sequence homology to known enzymes suggests a new type of enzyme.     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

Open and closed states of Mrlip1 DAG lipase revealed by molecular dynamics simulation

ABSTRACT

The lid and flap domains control the catalytic activity of lipase through the opening and closing motion. However, this gating mechanism of diacylglycerol (DAG) lipase is poorly understood due to the lack of 3D structures in open conformations. In this study, the opening and closing states of Mrlip1 DAG lipase are revealed by the homology modelling and molecular dynamic simulations. It was found that the active residues (Ser₁₇₁, His₂₈₁ and Asp₂₂₈) in the catalytic pocket of Mrlip1 DAG lipase are covered by the lid domain in the closed conformation, and exposed to the solvent in the open conformation. The role of residues Phe₂₇₈ and Gln₂₈₂ in the flap domain, as well as that of Thr₁₀₁ and Thr₁₀₇ in the lid domains are also identified in gating mechanism. The site-directed mutagenesis have been carried out to illustrate the putative alterations of enzyme specificity. Our results suggest that the substrate specificity is achieved by these two key residues Phe₂₇₈ and Gln₂₈₂, and the irreversible conversion from DAG to TAG (Triacylglycerol) lipase are enabled by the two-point mutations.     

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and temperature (5–85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.     

Tuning the substrate selectivity of meta-cleavage product hydrolase by domain swapping

meta-Cleavage product (MCP) hydrolases can catalyze relatively low reactive carbon–carbon bond hydrolysis of products, which are derived from the meta-cleavage of catechols. The strict substrate selectivity of MCP hydrolases attracts an interest to understand the determinants of substrate specificity. Compared with conventional site-directed mutagenesis, domain swapping is an effective strategy to explore substrate specificity due to the large-scale reorganization of three-dimensional structure. In the present study, the hybrid MCP hydrolases BphD_LidA and MfphA_LidD were constructed by exchanging the lid domain of two parental enzymes MfphA and BphD. The residues Gly130/Ala196 (MfphA) and Gly136/Ala211 (BphD) were selected as crossover points according to structural disruption score analysis and molecular dynamics simulations. It was shown that the hybrid enzymes exhibited similar substrate selectivity with the parent enzyme providing the lid domain. Docking studies suggested that the lid domain may play a key role in determining substrate specificity by reshaping the active pocket and modulating the orientation of the substrate.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.     

Solvent-induced lid opening in lipases: A molecular dynamics study

In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule.     

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures

A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (ΔL2-PML) was constructed by deleting residues 35-64 of PML. ΔL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ΔL2-PML did not. These results indicate that PML undergoes interfacial activation, while ΔL2-PML does not. The activities of ΔL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ΔL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML. DATABASE: Triacylglycerol lipase (EC 3.1.1.3).