A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.     

Trp-107 and Trp-253 Account for the Increased Steady State Fluorescence That Accompanies the Conformational Change in Human Pancreatic Triglyceride Lipase Induced by Tetrahydrolipstatin and Bile Salt

The conformation of a surface loop, the lid, controls activity of pancreatic triglyceride lipase (PTL) by moving from a position that sterically hinders substrate access to the active site into a new conformation that opens and configures the active site. Movement of the lid is accompanied by a large change in steady state tryptophan fluorescence. Although a change in the microenvironment of Trp-253, a lid residue, could account for the increased fluorescence, the mechanism and tryptophan residues have not been identified. To identify the tryptophan residues responsible for the increased fluorescence and to gain insight into the mechanism of lid opening and the structure of PTL in aqueous solution, we examined the effects of mutating individual tryptophan residues to tyrosine, alanine, or phenylalanine on lipase activity and steady state fluorescence. Substitution of tryptophans 86, 107, 253, and 403 reduced activity against tributyrin with the largest effects caused by substituting Trp-86 and Trp-107. Trp-107 and Trp-253 fluorescence accounts for the increased fluorescence emissions of PTL that is stimulated by tetrahydrolipstatin and sodium taurodeoxycholate. The largest contribution is from Trp-107. Contrary to the prediction from the crystal structure of PTL, Trp-107 is likely exposed to solvent. Both tetrahydrolipstatin and sodium taurodeoxycholate are required to produce the increased fluorescence in PTL. Alone, neither is sufficient. Colipase does not significantly influence the conformational changes leading to increased emission fluorescence. Thus, Trp-107 and Trp-253 contribute to the change in steady state fluorescence that is triggered by mixed micelles of inhibitor and bile salt. Furthermore, the results suggest that the conformation of PTL in solution differs significantly from the conformation in crystals.Lipases belong to a large gene family of proteins characterized by a common protein structure (1, 2). Included in this family are pancreatic triglyceride lipase (PTL,² triacylglycerol acylhydrolase, EC 3.1.1.3) and its close homologues pancreatic triglyceride lipase related proteins 1 and 2 (3). Not only do these pancreatic lipases have highly conserved primary structures, their x-ray crystal structures are essentially identical (4–6). Each contains two domains, a globular N-terminal domain consisting of an α/β hydrolase fold and a C-terminal domain consisting of a β-sandwich structure. A striking feature of these lipases and many others is the presence of a surface loop termed the lid domain. Together with the β5 loop and β9 loops of the N-terminal domain, the lid domain sterically hinders access of substrate to the active site. In this conformation, PTL cannot hydrolyze substrate, and the existence of another conformation was proposed (6).Subsequently, a second, open conformation of PTL was identified in studies of the crystal structure of the PTL-colipase complex (7, 8). In these studies, the investigators obtained crystals of the complex in the presence and absence of detergent and phospholipid mixed micelles. Without micelles, the lid domain remained in the same closed position as observed in the PTL structure even though colipase clearly bound to the C-terminal domain (8). With micelles, the lid domain and the β5 loop adopted new conformations (7). A large hinge movement of the lid moved the domain away from the active site to form new interactions with colipase. The lid movement opened and configured the active site to generate a conformation compatible with catalysis. Additionally, the movement exposed a large hydrophobic surface on the PTL-colipase complex, a surface that likely contributes to the anchoring of the complex on the substrate interface.Although x-ray crystallography studies clearly demonstrated two conformations of PTL and other lipases, these only provide a static picture of what may be the beginning and end of the process. The mechanism that triggers lid opening and the presence of intermediate conformations remains speculative. Initially, many assumed that a lipid-water interface triggered the conformational change (9). However, a number of studies using inhibitors, small angle neutron scattering, neutron diffraction, and monoclonal antibodies suggest that the lid can open in solution (10–14). In these studies, it was variously suggested that bile salt micelles and colipase or bile salt micelles alone were sufficient to trigger lid opening. The presence of a lipid substrate was not required.None of these studies addressed the relative contribution of bile salts and colipase to the lid opening. A recent paper described the use of electron paramagnetic resonance spectroscopy combined with site-directed spin labeling to monitor conformational changes in the PTL lid and to determine the effect of bile salts and colipase on lid opening (15). A cysteine was substituted for Asp-250 in the lid domain, and a paramagnetic probe was linked at that site. Using this method, the authors observed a mixture of closed and open conformations of the lid in the presence of bile salt micelles alone. Colipase by itself did not induce lid opening, but in the presence of bile salt micelles, colipase increased the relative concentration of PTL in the open conformation. Although the spin labeling did not have dramatic effects on the activity of the labeled PTL, it may not be benign. The presence of the probe may alter the kinetics of lid opening and may explain why a portion of PTL always stayed in the closed position.Another spectral method to follow conformation changes in proteins is fluorescence spectroscopy of native tryptophan. After systematically mutating the three tryptophans to alanine, investigators measured the binding of Thermomyces lanuginosus lipase and the mutants to mixed micelles of cis-parinaric acid and bile salt by fluorescence quenching and fluorescence resonance energy transfer (16). The measured values correlated with lid opening and depended on the presence of the single tryptophan in the lid. PTL shows a large increase in tryptophan fluorescence when incubated with a lipase inhibitor, tetrahydrolipstatin (THL), in the presence of bile salts (11). It was suggested, but not demonstrated, that the fluorescence change reflected movement of the lid domain. Because PTL contains seven tryptophan residues including one in the lid, Trp-253, the interpretation of this study is quite complicated. Another study monitoring time-resolved fluorescence of PTL and several tryptophan mutants demonstrated that Trp-30 makes a significant contribution to the tryptophan fluorescence of PTL (17). The lid tryptophan, Trp-253, had a low quantum yield and contributed considerably less to the overall tryptophan fluorescence. This report did not include investigations of PTL fluorescence in the presence of bile salts or colipase. Consequently, the assumption that the large increase in steady state fluorescence of PTL in the presence of THL and bile salt results from changes in the environment of the lid domain tryptophan remains unproven.To determine whether the increased tryptophan fluorescence of PTL in THL and bile saIt represents a conformational change in PTL, we measured the effect of tryptophan substitution mutations on the activity and intrinsic steady state fluorescence of PTL. Each of the seven tryptophans was mutated to tyrosine. Selected tryptophans were mutated to alanine or phenylalanine. Each mutant PTL was expressed and purified. We monitored the effect of bile salts, colipase, THL, and mixtures of these compounds on the steady state fluorescence of PTL.     

Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity

Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.     

Interfacial catalysis by lipases

We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8).

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.     

Digestive lipases: from three-dimensional structure to physiology

Human gastric lipase (HGL) is a lipolytic enzyme that is secreted by the chief cells located in the fundic part of the stomach. HGL plays an important role in lipid digestion, since it promotes the subsequent hydrolytic action of pancreatic lipase in duodenal lumen. Physiological studies have shown that HGL is able of acting not only in the highly acid stomach environment but also in the duodenum in synergy with human pancreatic lipase (HPL). Recombinant HGL (r-HGL) was expressed in the baculovirus/insect cell system in the form of an active protein with a molecular mass of 45 kDa. The specific activities of r-HGL were found to be similar to that of the native enzyme when tested on various triacylglycerol (TG) substrates. The 3-D structure of r-HGL was the first solved within the mammalian acid lipase family. This globular enzyme (379 residues) shows a new feature, different from the other known lipases structures, which consists of a core domain having the alpha/beta hydrolase fold and a cap domain including a putative 'lid' of 30 residues covering the active site of the lipase (closed conformation). HPL is the major lipolytic enzyme involved in the digestion of dietary TG. HPL is a 50 kDa glycoprotein which is directly secreted as an active enzyme. HPL was the first mammalian lipase to be solved structurally, and it revealed the presence of two structural domains: a large N-terminal domain (residues 1-336) and a smaller C-terminal domain (residues 337-449). The large N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site. A surface loop called the lid domain (C237-C261) covers the active site in the closed conformation of the lipase. The 3-D structure of the lipase-procolipase complex illustrates how the procolipase might anchor the lipase at the interface in the presence of bile salts: procolipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite site of its lipase-binding domain. These hydrophobic tips help to bring N-terminal domain into close conformation with the interface where the opening of the lid domain probably occurs. As a result of all these conformational changes, the open lid and the extremities of the procolipase form an impressive continuous hydrophobic plateau, extending over more than 50 A. This surface might able to interact strongly with a lipid-water interface. The biochemical, histochemical and clinical studies as well as the 3-D structures obtained will be a great help for a better understanding of the structure-function relationships of digestive lipases.     

Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants

Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.     

X-ray Crystallographic and MD Simulation Studies on the Mechanism of Interfacial Activation of a Family I.3 Lipase with Two Lids

The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two α-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 Å resolution in the presence of Ca²⁺ and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca²⁺-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca²⁺ and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.     

In vitro lipolysis by human pancreatic lipase is specifically abolished by its inactive forms

In human adults, the enzymatic hydrolysis of dietary fat along the digestive tract is sequentially catalyzed by two main enzymes, human gastric lipase (HGL) and human pancreatic lipase (HPL). Both a chemically inhibited form of HPL as well as an inactive HPL mutant with a glycine residue substituted for its catalytic serine were found to be strong inactivators of HPL activity. In the presence of bile salts, this inhibition was clearly due to competition for colipase. We established that the chemically inhibited HPL, probably in its open conformation, had a much greater affinity for colipase than the closed native form of HPL. These inhibitory effects are quite substantial, because a 0.2-M excess of the chemically inhibited HPL form relative to HPL reduced the catalytic lipolytic activity by 50% in the presence of an equimolar amount of colipase.     

Lid closure dynamics of porcine pancreatic lipase in aqueous solution

BackgroundPancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water.MethodsMolecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated.ResultsThe lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around β5- and β9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation.ConclusionsPPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure.General significanceConformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.     

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.     

Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.     

Human pancreatic lipase-related protein 2 is a galactolipase

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.     

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.     

Resolved conformational states of spin-labeled Ca-ATPase during the enzymatic cycle.

We have used frequency- and time-resolved electron paramagnetic resonance (EPR) to study the effects of substrate on the nanosecond conformational dynamics of the Ca-ATPase of sarcoplasmic reticulum, as detected by an iodoacetamide spin label (IASL) attached covalently to the enzyme. We confirm previous results [Coan, C. (1983) Biochemistry 22, 5826] showing that this probe is less rotationally mobile following the addition of nucleotides (ADP, AMPPNP, ATP) and that the shape of the spectrum suggests the presence of two components. We used two approaches to enhance EPR resolution in order to resolve the spectral components and their corresponding conformational states. First, to improve resolution in the frequency (spectral) domain, we used perdeuterated IASL, which results in narrower line widths. Digital spectral analysis resolves the EPR spectrum into two components, one that is indistinguishable from the spectrum observed in the absence of ligands and another that indicates more restricted probe motions, suggesting a distinct conformation of the labeled protein. Additions of substrate ligands appear to change only the mole fractions of the two components. The mole fraction of the restricted component (fR) was 0 in the absence of ligands, but increased to about 0.5 in the presence of saturating concentrations of AMPPNP and Ca2+. In general, ATP and its analogs increase fR, with larger effects observed in the presence of Ca. However, calcium has no effect by itself (fR = 0). Both monovanadate and decavanadate increase fR, but the formation of a covalent phosphoenzyme from inorganic phosphate (E2-P) had no effect (fR = 0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrated analysis of the conformation of a protein-linked spin label by crystallography,EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.     

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.     

The open lid mediates pancreatic lipase function

Pancreatic triglyceride lipase (PTL) and the homologous pancreatic lipase related protein 2 (PLRP2) provide a unique opportunity to understand the molecular mechanism of lipolysis. They differ in substrate specificity, sensitivity to bile salts, and colipase dependence despite their close amino acid and tertiary structure identity. One important structure, present in both lipases, is the lid which occupies different positions in the inactive and active forms of PTL. We investigated the role of the lid in lipase function by site-specific mutagenesis. By exchanging the lids between PTL and PLRP2, we created two chimeric lipases. Additionally, we made multiple substitution mutations in the PTL lid. PLRP2 with the PTL lid had kinetic properties similar to PLRP2. PTL with the PLRP2 lid was greatly impaired and had no activity at micellar bile salt concentrations even in the presence of colipase. Both chimeras showed interfacial activation suggesting that the closed lid position was maintained. A series of substitution mutations were made in positions Arg257 and Asp258. These mutations demonstrated the importance of these two residues to maintaining the normal activity, triglyceride acyl chain specificity, and colipase interaction of PTL. The preserved interfacial activation in the chimeras, the similar crystal structure of the two lids in the closed position, and the importance of Arg257 and Asp258 in mediating the open conformation of the lid argue that the position of the open lid influences the differences in activity against triglycerides, in sensitivity to bile salts, and in colipase dependence between PTL and PLRP2.     

Rapid exchange of pancreatic lipase between triacylglycerol droplets

Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.     

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).     

Combining EPR with Fluorescence Spectroscopy to Monitor Conformational Changes at the Myosin Nucleotide Pocket

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WT⋅SLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin⋅myosin⋅diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins.