Roussel A

References (15)

Title : Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3\/Tli3 T6SS Effector\/Immunity Complex Determined Using an AlphaFold2 Predicted Model - Le_2023_Int.J.Mol.Sci_24_1740
Author(s) : Le TTH , Kellenberger C , Boyer M , Santucci P , Flaugnatti N , Cascales E , Roussel A , Canaan S , Journet L , Cambillau C
Ref : Int J Mol Sci , 24 :1740 , 2023
Abstract : The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3-an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3(AIEC) has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3(AIEC). Tli3(AIEC) binds Tle3 in a 1:1 stoichiometric ratio. Tle3(AIEC), Tli3(AIEC) and the Tle3(AIEC)-Tli3(AIEC) complex were purified and subjected to crystallization. The Tle3(AIEC)-Tli3(AIEC) complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3(AIEC) exhibits an alpha/beta-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3(AIEC) displays a new fold of three stacked beta-sheets and a protruding loop that inserts in Tle3(AIEC)catalytic crevice. We showed, experimentally, that Tle3(AIEC) interacts with the VgrG (AIEC) cargo protein and AlphaFold2 prediction of the VgrG(AIEC)-Tle3(AIEC) complex reveals a strong interaction between the VgrG(AIEC) C-terminus adaptor and Tle3(AIEC) N-terminal loop.
ESTHER : Le_2023_Int.J.Mol.Sci_24_1740
PubMedSearch : Le_2023_Int.J.Mol.Sci_24_1740
PubMedID: 36675258
Gene_locus related to this paper: ecoli-T6SS.TLE3

Title : A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography - de Wijn_2019_IUCrJ_6_454
Author(s) : de Wijn R , Hennig O , Roche J , Engilberge S , Rollet K , Fernandez-Millan P , Brillet K , Betat H , Morl M , Roussel A , Girard E , Mueller-Dieckmann C , Fox GC , Olieric V , Gavira JA , Lorber B , Sauter C
Ref : IUCrJ , 6 :454 , 2019
Abstract : Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.
ESTHER : de Wijn_2019_IUCrJ_6_454
PubMedSearch : de Wijn_2019_IUCrJ_6_454
PubMedID: 31098026
Gene_locus related to this paper: humla-1lipa

Title : Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors - Mateos-Diaz_2017_Methods.Enzymol_583_279
Author(s) : Mateos-Diaz E , Amara S , Roussel A , Longhi S , Cambillau C , Carriere F
Ref : Methods Enzymol , 583 :279 , 2017
Abstract : Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.
ESTHER : Mateos-Diaz_2017_Methods.Enzymol_583_279
PubMedSearch : Mateos-Diaz_2017_Methods.Enzymol_583_279
PubMedID: 28063495

Title : A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery - Flaugnatti_2016_Mol.Microbiol_99_1099
Author(s) : Flaugnatti N , Le TT , Canaan S , Aschtgen MS , Nguyen VS , Blangy S , Kellenberger C , Roussel A , Cambillau C , Cascales E , Journet L
Ref : Molecular Microbiology , 99 :1099 , 2016
Abstract : The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.
ESTHER : Flaugnatti_2016_Mol.Microbiol_99_1099
PubMedSearch : Flaugnatti_2016_Mol.Microbiol_99_1099
PubMedID: 26714038
Gene_locus related to this paper: ecolx-h4unx0

Title : A Cutinase from Trichoderma reesei with a Lid-Covered Active Site and Kinetic Properties of True Lipases - Roussel_2014_J.Mol.Biol_426_3757
Author(s) : Roussel A , Amara S , Nyyssola A , Mateos-Diaz E , Blangy S , Kontkanen H , Westerholm-Parvinen A , Carriere F , Cambillau C
Ref : Journal of Molecular Biology , 426 :3757 , 2014
Abstract : Cutinases belong to the alpha/beta-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded beta-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as beta-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.
ESTHER : Roussel_2014_J.Mol.Biol_426_3757
PubMedSearch : Roussel_2014_J.Mol.Biol_426_3757
PubMedID: 25219509
Gene_locus related to this paper: hypjq-g0rh85

Title : Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study - Miled_2003_Biochemistry_42_11587
Author(s) : Miled N , Roussel A , Bussetta C , Berti-Dupuis L , Riviere M , Buono G , Verger R , Cambillau C , Canaan S
Ref : Biochemistry , 42 :11587 , 2003
Abstract : The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.
ESTHER : Miled_2003_Biochemistry_42_11587
PubMedSearch : Miled_2003_Biochemistry_42_11587
PubMedID: 14529268

Title : Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor - Roussel_2002_J.Biol.Chem_277_2266
Author(s) : Roussel A , Miled N , Berti-Dupuis L , Riviere M , Spinelli S , Berna P , Gruber V , Verger R , Cambillau C
Ref : Journal of Biological Chemistry , 277 :2266 , 2002
Abstract : Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-A resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Here we report on the open structure of the recombinant dog gastric lipase (r-DGL) at 2.7-A resolution in complex with the undecyl-butyl (C11Y4) phosphonate inhibitor. HGL and r-DGL show 85.7% amino acid sequence identity, which makes it relevant to compare the forms from two different species. The open r-DGL structure confirms the previous description of the HGL catalytic triad (Ser(153), His(353), and Asp(324)) with the catalytic serine buried and an oxyanion hole (NH groups of Gln(154) and Leu(67)). In r-DGL, the binding of the C11Y4 phosphonate inhibitor induces part of the cap domain, the lid, to roll over the enzyme surface and to expose a catalytic crevice measuring approximately 20 x 20 x 7 A(3). The C11Y4 phosphonate fits into this crevice, and a molecule of beta-octyl glucoside fills up the crevice. The C11Y4 phosphonate inhibitor and the detergent molecule suggest a possible binding mode for the natural substrates, the triglyceride molecules.
ESTHER : Roussel_2002_J.Biol.Chem_277_2266
PubMedSearch : Roussel_2002_J.Biol.Chem_277_2266
PubMedID: 11689574
Gene_locus related to this paper: canfa-1lipg

Title : Digestive lipases: from three-dimensional structure to physiology - Miled_2000_Biochimie_82_973
Author(s) : Miled N , Canaan S , Dupuis L , Roussel A , Riviere M , Carriere F , de Caro A , Cambillau C , Verger R
Ref : Biochimie , 82 :973 , 2000
Abstract : 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.
ESTHER : Miled_2000_Biochimie_82_973
PubMedSearch : Miled_2000_Biochimie_82_973
PubMedID: 11099794

Title : Gastric lipase: crystal structure and activity - Canaan_1999_Biochim.Biophys.Acta_1441_197
Author(s) : Canaan S , Roussel A , Verger R , Cambillau C
Ref : Biochimica & Biophysica Acta , 1441 :197 , 1999
Abstract : Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We have solved the structure of recombinant human gastric lipase at 3.0 A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain, belonging to the alpha/beta hydrolase fold family, and an extrusion domain. It possesses a classical catalytic triad (Ser 153, His 353, Asp 324) and an oxyanion hole (NH groups of Gln 154 and Leu 67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 30 residues, which can be defined as a lid. Its displacement is necessary for the substrates to access the active site. A phosphonate inhibitor was positioned in the active site which clearly suggests the location of the hydrophobic substrate binding site.
ESTHER : Canaan_1999_Biochim.Biophys.Acta_1441_197
PubMedSearch : Canaan_1999_Biochim.Biophys.Acta_1441_197
PubMedID: 10570247
Gene_locus related to this paper: human-LIPF

Title : Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest - Roussel_1999_J.Biol.Chem_274_16995
Author(s) : Roussel A , Canaan S , Egloff MP , Riviere M , Dupuis L , Verger R , Cambillau C
Ref : Journal of Biological Chemistry , 274 :16995 , 1999
Abstract : Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active despite the highly acidic stomach environment. We report here the structure of recombinant human gastric lipase at 3.0-A resolution, the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists of a core domain belonging to the alpha/beta hydrolase-fold family and a "cap" domain, which is analogous to that present in serine carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of Gln-154 and Leu-67). Four N-glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.
ESTHER : Roussel_1999_J.Biol.Chem_274_16995
PubMedSearch : Roussel_1999_J.Biol.Chem_274_16995
PubMedID: 10358049
Gene_locus related to this paper: human-LIPF

Title : Structure and activity of rat pancreatic lipase-related protein 2 - Roussel_1998_J.Biol.Chem_273_32121
Author(s) : Roussel A , Yang Y , Ferrato F , Verger R , Cambillau C , Lowe M
Ref : Journal of Biological Chemistry , 273 :32121 , 1998
Abstract : The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 A structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a beta-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.
ESTHER : Roussel_1998_J.Biol.Chem_273_32121
PubMedSearch : Roussel_1998_J.Biol.Chem_273_32121
PubMedID: 9822688
Gene_locus related to this paper: ratno-4plip

Title : Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations - Roussel_1998_Proteins_32_523
Author(s) : Roussel A , De Caro J , Bezzine S , Gastinel L , de Caro A , Carriere F , Leydier S , Verger R , Cambillau C
Ref : Proteins , 32 :523 , 1998
Abstract : Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.
ESTHER : Roussel_1998_Proteins_32_523
PubMedSearch : Roussel_1998_Proteins_32_523
PubMedID: 9726421
Gene_locus related to this paper: canfa-1plip

Title : Structural basis for the substrate selectivity of pancreatic lipases and some related proteins - Carriere_1998_Biochim.Biophys.Acta_1376_417
Author(s) : Carriere F , Withers-Martinez C , van Tilbeurgh H , Roussel A , Cambillau C , Verger R
Ref : Biochimica & Biophysica Acta , 1376 :417 , 1998
Abstract : The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.
ESTHER : Carriere_1998_Biochim.Biophys.Acta_1376_417
PubMedSearch : Carriere_1998_Biochim.Biophys.Acta_1376_417
PubMedID: 9805004

Title : An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure - Bezzine_1998_Chem.Phys.Lipids_93_103
Author(s) : Bezzine S , Roussel A , De Caro J , Gastinel L , de Caro A , Carriere F , Leydier S , Verger R , Cambillau C
Ref : Chemistry & Physic of Lipids , 93 :103 , 1998
Abstract : Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.
ESTHER : Bezzine_1998_Chem.Phys.Lipids_93_103
PubMedSearch : Bezzine_1998_Chem.Phys.Lipids_93_103
PubMedID: 9720253

Title : Lipoprotein lipase. Molecular model based on the pancreatic lipase x-ray structure: consequences for heparin binding and catalysis - van Tilbeurgh_1994_J.Biol.Chem_269_4626
Author(s) : van Tilbeurgh H , Roussel A , Lalouel JM , Cambillau C
Ref : Journal of Biological Chemistry , 269 :4626 , 1994
Abstract : Lipoprotein lipase and pancreatic lipase have about 30% sequence identity, suggesting a similar tertiary fold. Three-dimensional models of lipoprotein lipase were constructed, based upon two recently determined x-ray crystal structures of pancreatic lipase, in which the active site was in an open and closed conformation, respectively. These models allow us to propose a few hypotheses on the structural determinants of lipoprotein lipase which are responsible for heparin binding, dimer formation, and phospholipase activity. The folding of the protein assembles a number of positive charge clusters at the back of the molecule, opposite the active site. These clusters probably form the heparin binding site, as confirmed by recent site-directed mutagenesis experiments. The active sites of lipoprotein lipase and pancreatic lipase look very similar, except for the lid (a surface loop covering the catalytic serine in the inactive state). A different open (active) conformation of the lid in both enzymes may be responsible for their differing substrate specificities. Predictions of the nature of the lipoprotein lipase dimer remain elusive, although our model enabled us to propose a few possibilities.
ESTHER : van Tilbeurgh_1994_J.Biol.Chem_269_4626
PubMedSearch : van Tilbeurgh_1994_J.Biol.Chem_269_4626
PubMedID: 8308035