Dynamics of cholesterol exchange in the oxysterol binding protein family

The oxysterol-binding protein-related protein (ORP) family is essential to sterol transfer and sterol-dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol-bound states. In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.     

Theoretical investigation of the dynamics of the active site lid in Rhizomucor miehei lipase.

Interfacial activation of Rhizomucor miehei lipase is accompanied by a hinge-type motion of a single helix (residues 83-94) that acts as a lid over the active site. Activation of the enzyme involves the displacement of the lid to expose the active site, suggesting that the dynamics of the lid could be of mechanistic and kinetic importance. To investigate possible activation pathways and to elucidate the effect of a hydrophobic environment (as would be provided by a lipid membrane) on the lid opening, we have applied molecular dynamics and Brownian dynamics techniques. Our results indicate that the lipase activation is enhanced in a hydrophobic environment. In nonpolar low-dielectric surroundings, the lid opens in approximately 100 ns in the BD simulations. In polar high-dielectric (aqueous) surroundings, the lid does not always open up in simulations of up to 900 ns duration, but it does exhibit some gating motion, suggesting that the enzyme molecule may exist in a partially active form before the catalytic reaction. The activation is controlled by the charged residues ARG86 and ASP91. In the inactive conformation, ASP91 experiences repulsive forces and pushes the lid toward the open conformation. Upon activation ARG86 approaches ASP61, and in the active conformation, these residues form a salt bridge that stabilizes the open conformation.     

3-D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.     

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.     

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).     

Crystal structure analysis,covalent docking,and molecular dynamics calculations reveal a conformational switch in PhaZ7 PHB depolymerase

An open and a closed conformation of a surface loop in PhaZ7 extracellular poly(3‐hydroxybutyrate) depolymerase were identified in two high‐resolution crystal structures of a PhaZ7 Y105E mutant. Molecular dynamics (MD) simulations revealed high root mean square fluctuations (RMSF) of the 281–295 loop, in particular at residue Asp289 (RMSF 7.62 Å). Covalent docking between a 3‐hydroxybutyric acid trimer and the catalytic residue Ser136 showed that the binding energy of the substrate is significantly more favorable in the open loop conformation compared to that in the closed loop conformation. MD simulations with the substrate covalently bound depicted 1 Å RMSF higher values for the residues 281–295 in comparison to the apo (substrate‐free) form. In addition, the presence of the substrate in the active site enhanced the ability of the loop to adopt a closed form. Taken together, the analysis suggests that the flexible loop 281–295 of PhaZ7 depolymerase can act as a lid domain to control substrate access to the active site of the enzyme. Proteins 2017; 85:1351–1361. © 2017 Wiley Periodicals, Inc.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

In silico and empirical approaches toward understanding the structural adaptation of the alkaline-stable lipase KV1 from Acinetobacter haemolyticus

Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.     

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Different Inward and Outward Conduction Mechanisms in NaVMs Suggested by Molecular Dynamics Simulations

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. Here, we use molecular dynamics (MD) simulations with an applied membrane potential to investigate the ion flux of bacterial sodium channel Na_VMs. 5.9 µs simulations with 500 mM NaCl suggest different mechanisms for inward and outward flux. The predicted inward conductance rate of ∼27±3 pS, agrees with experiment. The estimated outward conductance rate is 15±3 pS, which is considerably lower. Comparing inward and outward flux, the mean ion dwell time in the selectivity filter (SF) is prolonged from 13.5±0.6 ns to 20.1±1.1 ns. Analysis of the Na⁺ distribution revealed distinct patterns for influx and efflux events. In 32.0±5.9% of the simulation time, the E53 side chains adopted a flipped conformation during outward conduction, whereas this conformational change was rarely observed (2.7±0.5%) during influx. Further, simulations with dihedral restraints revealed that influx is less affected by the E53 conformational flexibility. In contrast, during outward conduction, our simulations indicate that the flipped E53 conformation provides direct coordination for Na⁺. The free energy profile (potential of mean force calculations) indicates that this conformational change lowers the putative barriers between sites S_CEN and S_HFS during outward conduction. We hypothesize that during an action potential, the increased Na⁺ outward transition propensities at depolarizing potentials might increase the probability of E53 conformational changes in the SF. Subsequently, this might be a first step towards initiating slow inactivation.     

Holo‐BtuF stabilizes the open conformation of the vitamin B12 ABC transporter BtuCD

While there is evidence that other ABC transporters can tell between empty and loaded substrate binding protein, reconstitution experiments suggest otherwise for the Escherichia coli vitamin B12 importer BtuCD‐F. Here, we address the question of BtuCD‐F substrate sensitivity in a combined protein–protein docking and molecular dynamics simulation approach. Starting from the BtuCD and holo‐BtuF crystal structures, we model two holo‐BtuCD‐F docking complexes differing by a 180° orientation of BtuF. One of these is similar to the apo‐BtuCD‐F crystal structure. Both docking complexes were embedded in a lipid/water environment to investigate their dynamics and BtuCD's conformational response to the presence and absence of BtuF, vitamin B12, and Mg‐ATP in a series of 28 independent MD simulations. We find holo‐BtuF stabilizing the open conformation of BtuCD, whereas the transporter begins to close again when BtuF or vitamin B12 is removed—suggesting BtuCD‐F is capable of substrate sensitivity. We identified BtuC transmembrane helices 3 and 5, the L‐loops and the adjacent helices comprised of BtuC residues 170–180 as hotspots of conformational change. We propose the latter to act as substrate sensors. BtuF‐Trp44 appears to act as a lid on the vitamin B12 binding cleft in BtuF X‐ray structures and protrudes into the BtuCD transport channel in one of our simulations, which might represent an initial step in vitamin B12 uptake. On an average, we observe subunit motions where the nucleotide binding domains approach each other while the transmembrane domains display an opening trend toward the periplasm. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Simulation of the substrate cavity dynamics of quercetinase

Molecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu-containing dioxygenase known so far. It catalyses the breakage of the O-heterocycle of flavonols. The substrates occupy a shallow and overall hydrophobic cavity proximal to the metal centre of the homo-dimeric enzyme. The linker connecting the C-terminal and N-terminal domains in the monomer is partly disordered in the crystal structure and part of it forms a flexible lid at the entrance of the substrate cavity. This loop has been tentatively assigned a role in the enzyme mechanism: it helps lock the substrate into place. The dynamics of this loop has been investigated by MD simulation. The initial coordinates were taken from the crystal structure of 2,3QD in the presence of the substrate kaempferol (KMP). After equilibration and simulation over 7.2ns the substrate was removed and another equilibration and simulation of 7.2ns was performed. The results show that the structures of the free enzyme as well as of the enzyme-substrate complex are stable in MD simulation. The linker shows strongly enhanced mobility in the loop region that is close to the entrance to the substrate cavity (residues 154-169). Movement of the loop takes place on a timescale of 5-10ns. To confirm the conclusions about the loop dynamics drawn from the 7.2ns simulation, the simulation was extended with another 8ns. When substrate binds into the cavity the loop orders remarkably, although mobility is retained by residues 155-158. Some regions of the loop (residues 154-160 and 164-176) move over a considerable distance and approach the substrate closely, reinforcing the idea that they lock the substrate in the substrate cavity. The enthalpic component of the interaction of the loop with the protein and the KMP appears to favour the locking of the substrate. Two water molecules were found immobilised in the cavity, one of which exhibited rotation on the picosecond timescale. When the substrate is removed, the empty cavity fills up with water within 200ps.     

Long-chain fatty acyl-CoA esters induce lipase activation in the absence of a water-lipid interface

In most lipases a mobile element or lid domain covers the catalytic site of the enzyme and the lid opening event, which usually proceed at a lipid-water interface, is required to form the catalytically competent lipase. We report here a noticeable increase in activity of two fungal lipases assayed in aqueous solution in absence of any interface when adding submicellar concentrations of amphipathic physiological molecules like long-chain acyl-CoAs. The catalytic activity was dramatically dependent on the acyl chain length of the amphiphile and could be related with a lid-opening process. Our data support that lipase activation can be triggered in the absence of a well-defined interface, and stresses the notion that other non-aggregated amphipathic constituents of the local microenvironment can act as putative regulators of lipase activity.     

Study of Thermomyces lanuginosa Lipase in the Presence of Tributyrylglycerol and Water

The Thermomyces lanuginosa lipase has been extensively studied in industrial and biotechnological research because of its potential for triacylglycerol transformation. This protein is known to catalyze both hydrolysis at high water contents and transesterification in quasi-anhydrous conditions. Here, we investigated the Thermomyces lanuginosa lipase structure in solution in the presence of a tributyrin aggregate using 30 ns molecular-dynamics simulations. The water content of the active-site groove was modified between the runs to focus on the protein-water molecule interactions and their implications for protein structure and protein-lipid interactions. The simulations confirmed the high plasticity of the lid fragment and showed that lipid molecules also bind to a secondary pocket beside the lid. Together, these results strongly suggest that the lid plays a role in the anchoring of the protein to the aggregate. The simulations also revealed the existence of a polar channel that connects the active-site groove to the outside solvent. At the inner extremity of this channel, a tyrosine makes hydrogen bonds with residues interacting with the catalytic triad. This system could function as a pipe (polar channel) controlled by a valve (the tyrosine) that could regulate the water content of the active site.     

Crystal structure of N-acetyl-gamma-glutamyl-phosphate reductase from Mycobacterium tuberculosis in complex with NADP(+)

The enzyme N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductive dephosphorylation of N-acetyl-gamma-glutamyl-phosphate to N-acetylglutamate-gamma-semialdehyde. This reaction is part of the arginine biosynthetic pathway that is essential for some microorganisms and plants, in particular, for Mycobacterium tuberculosis (Mtb). The structures of apo MtbAGPR in the space groups P2(1)2(1)2(1) and C2 and the structure of MtbAGPR bound to the cofactor NADP(+) have been solved and analyzed. Each MtbAGPR subunit consists of alpha/beta and alpha+beta domains; NADP(+) is bound in the cleft between them. The hydrogen bonds and hydrophobic contacts between the enzyme and cofactor have been examined. Comparison of the apo and the bound enzyme structures has revealed a conformational change in MtbAGPR upon NADP(+) binding. Namely, a loop (Leu88 to His92) moves more than 5 A to confine sterically the cofactor's adenine moiety in a hydrophobic pocket. To identify the catalytically important residues in MtbAGPR, a docking of the substrate to the enzyme has been performed using the present structure of the MtbAGPR/NADP(+) complex. It reveals that residues His217 and His219 could form hydrogen bonds with the docked substrate. In addition, an ion pair could form between the substrate phosphate group and the guanidinium group of Arg114. These interactions optimally place and orient the substrate for subsequent nucleophilic attack by Cys158 on the substrate gamma-carboxyl group. His219 is the most probable general base to accept a proton from Cys158 and an adjacent ion pair interaction with the side-chain carboxyl group of Glu222 could help to stabilize the resulting positive charge on His219. For this catalytic triad to function efficiently it requires a small conformational change of the order of 1 A in the loop containing His217 and His219; this could easily result from the substrate binding.     

Molecular dynamics simulations on discoidal HDL particles suggest a mechanism for rotation in the apo A-I belt model

Apolipoprotein A-I (apo A-I) is the major protein component of high-density lipoprotein (HDL) particles. Elevated levels of HDL in the bloodstream have been shown to correlate strongly with a reduced risk factor for atherosclerosis. Molecular dynamics simulations have been carried out on three separate model discoidal high-density lipoprotein particles (HDL) containing two monomers of apo A-I and 160 molecules of palmitoyloleoylphosphatidylcholine (POPC), to a time-scale of 1ns. The starting structures were on the basis of previously published molecular belt models of HDL consisting of the lipid-binding C-terminal domain (residues 44-243) wrapped around the circumference of a discoidal HDL particle. Subtle changes between two of the starting structures resulted in significantly different behavior during the course of the simulation. The results provide support for the hypothesis of Segrest et al. that helical registration in the molecular belt model of apo A-I is modulated by intermolecular salt bridges. In addition, we propose an explanation for the presence of proline punctuation in the molecular belt model, and for the presence of two 11-mer helical repeats interrupting the otherwise regular pattern of 22-mer helical repeats in the lipid-binding domain of apo A-I.     

Molecular origins of asymmetric proton conduction in the influenza M2 channel

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance, but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction, Asp44 traps the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. Trp41 adds to the barrier by weakly H-bonding to potential H⁺ acceptors. Finally, for charged His, the H₃O⁺ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce an outward current appears to arise from its ability to bias H₃O⁺ and the waters around it toward more H-outward orientations.     

Unlocking the mystery behind the activation phenomenon of T1 lipase: a molecular dynamics simulations approach

The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable.