Cambillau C

General

Full name : Cambillau Christian

First name : Christian

Mail : Architecture et Fonction des Macromolecules Biologiques, Aix Marseille Universite UMR7257, Centre National de la Recherche Scientifique, 13288 Marseille Cedex 09

Zip Code :

City :

Country : France

Email : cambillau@afmb.univ-mrs.fr

Phone :

Fax :

Website :

Directory :

References (47)

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 : Biochemical and Structural Characterization of TesA, a Major Thioesterase Required for Outer-Envelope Lipid Biosynthesis in Mycobacterium tuberculosis - Nguyen_2018_J.Mol.Biol_430_5120
Author(s) : Nguyen PC , Nguyen VS , Martin BP , Fourquet P , Camoin L , Spilling CD , Cavalier JF , Cambillau C , Canaan S
Ref : Journal of Molecular Biology , 430 :5120 , 2018
Abstract : With the high number of patients infected by tuberculosis and the sharp increase of drug-resistant tuberculosis cases, developing new drugs to fight this disease has become increasingly urgent. In this context, analogs of the naturally occurring enolphosphates Cyclipostins and Cyclophostin (CyC analogs) offer new therapeutic opportunities. The CyC analogs display potent activity both in vitro and in infected macrophages against several pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium abscessus. Interestingly, these CyC inhibitors target several enzymes with active-site serine or cysteine residues that play key roles in mycobacterial lipid and cell wall metabolism. Among them, TesA, a putative thioesterase involved in the synthesis of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), has been identified. These two lipids (PDIM and PGL) are non-covalently bound to the outer cell wall in several human pathogenic mycobacteria and are important virulence factors. Herein, we used biochemical and structural approaches to validate TesA as an effective pharmacological target of the CyC analogs. We confirmed both thioesterase and esterase activities of TesA, and showed that the most active inhibitor CyC17 binds covalently to the catalytic Ser104 residue leading to a total loss of enzyme activity. These data were supported by the X-ray structure, obtained at a 2.6-A resolution, of a complex in which CyC17 is bound to TesA. Our study provides evidence that CyC17 inhibits the activity of TesA, thus paving the way to a new strategy for impairing the PDIM and PGL biosynthesis, potentially decreasing the virulence of associated mycobacterial species.
ESTHER : Nguyen_2018_J.Mol.Biol_430_5120
PubMedSearch : Nguyen_2018_J.Mol.Biol_430_5120
PubMedID: 30292819
Gene_locus related to this paper: myctu-yt28

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 : Biochemical and structural characterization of non-glycosylated Yarrowia lipolytica LIP2 lipase - Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
Author(s) : Aloulou A , Benarouche A , Puccinelli D , Spinelli S , Cavalier JF , Cambillau C , Carriere F
Ref : Eur.J.Lipid.Sci.Tech , 115 :429 , 2013
Abstract : The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) is assumed to be a good drug candidate for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. Understanding and improving its biochemical properties are essential for its oral administration. YLLIP2 is a highly glycosylated protein, with glycan chains accounting for about 13% of the molecular mass of the native protein. Two potential N-glycosylation sites (N113IS and N134NT) can be identified from YLLIP2 amino acid sequence. YLLIP2 mutants with single (N113Q or N134Q) or combined (N113Q/N134Q) substitutions of these glycosylation sites were expressed in the yeast Pichia pastoris, purified and characterized. Lipase specific activity and adsorption at the lipidwater interface were found to be decreased in the absence of N-glycosylation. It was thus shown that the glycosylated enzyme had a better ability to bind and penetrate a DLPC monolayer than the non-glycosylated N113Q/N134Q mutant. Comparison of wild-type glycosylated and non-glycosylated YLLIP2 shows that the N-glycosylation clearly contributes to the high stability of YLLIP2 in the presence of pepsin in vitro, and to a lower extent in the presence of chymotrypsin. The X-ray structure of the YLLIP2 N113Q/N134Q double mutant was obtained at 2.6 angstrom resolution and was found to be identical to that of wild-type YLLIP2, with the lid in a closed conformation. Glycosylation is therefore not essential for a proper folding of YLLIP2. Practical applications: The LIP2 lipase from the yeast Yarrowia lipolytica is one of the most active lipases identified so far. Among the various applications envisioned for this enzyme, it seems particularly well adapted for enzyme replacement therapy in patients with pancreatic exocrine insufficiency. It is active and stable at low pH values, resistant to bile salts, and its glycosylation allows a high resistance to pepsin. All these properties are important for developing the oral administration of digestive enzymes used as drugs.
ESTHER : Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
PubMedSearch : Aloulou_2013_Eur.J.Lipid.Sci.Tech_115_429
PubMedID:
Gene_locus related to this paper: yarli-lip2

Title : Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation - Eydoux_2008_Biochemistry_47_9553
Author(s) : Eydoux C , Spinelli S , Davis TL , Walker JR , Seitova A , Dhe-Paganon S , de Caro A , Cambillau C , Carriere F
Ref : Biochemistry , 47 :9553 , 2008
Abstract : 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.
ESTHER : Eydoux_2008_Biochemistry_47_9553
PubMedSearch : Eydoux_2008_Biochemistry_47_9553
PubMedID: 18702514
Gene_locus related to this paper: human-PNLIPRP2

Title : LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis - Sulzenbacher_2006_EMBO.J_25_1436
Author(s) : Sulzenbacher G , Canaan S , Bordat Y , Neyrolles O , Stadthagen G , Roig-Zamboni V , Rauzier J , Maurin D , Laval F , Daffe M , Cambillau C , Gicquel B , Bourne Y , Jackson M
Ref : EMBO Journal , 25 :1436 , 2006
Abstract : Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U-shaped beta-half-barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram-negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope.
ESTHER : Sulzenbacher_2006_EMBO.J_25_1436
PubMedSearch : Sulzenbacher_2006_EMBO.J_25_1436
PubMedID: 16541102

Title : Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis - Canaan_2005_FEBS.Lett_579_215
Author(s) : Canaan S , Sulzenbacher G , Roig-Zamboni V , Scappuccini-Calvo L , Frassinetti F , Maurin D , Cambillau C , Bourne Y
Ref : FEBS Letters , 579 :215 , 2005
Abstract : With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.
ESTHER : Canaan_2005_FEBS.Lett_579_215
PubMedSearch : Canaan_2005_FEBS.Lett_579_215
PubMedID: 15620716

Title : Automated expression and solubility screening of His-tagged proteins in 96-well format - Vincentelli_2005_Anal.Biochem_346_77
Author(s) : Vincentelli R , Canaan S , Offant J , Cambillau C , Bignon C
Ref : Analytical Biochemistry , 346 :77 , 2005
Abstract : A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time.
ESTHER : Vincentelli_2005_Anal.Biochem_346_77
PubMedSearch : Vincentelli_2005_Anal.Biochem_346_77
PubMedID: 16168382

Title : Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family - Canaan_2004_Eur.J.Biochem_271_3953
Author(s) : Canaan S , Maurin D , Chahinian H , Pouilly B , Durousseau C , Frassinetti F , Scappuccini-Calvo L , Cambillau C , Bourne Y
Ref : European Journal of Biochemistry , 271 :3953 , 2004
Abstract : The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.
ESTHER : Canaan_2004_Eur.J.Biochem_271_3953
PubMedSearch : Canaan_2004_Eur.J.Biochem_271_3953
PubMedID: 15373841
Gene_locus related to this paper: myctu-Rv1399c

Title : High-throughput automated refolding screening of inclusion bodies - Vincentelli_2004_Protein.Sci_13_2782
Author(s) : Vincentelli R , Canaan S , Campanacci V , Valencia C , Maurin D , Frassinetti F , Scappucini-Calvo L , Bourne Y , Cambillau C , Bignon C
Ref : Protein Science , 13 :2782 , 2004
Abstract : One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.
ESTHER : Vincentelli_2004_Protein.Sci_13_2782
PubMedSearch : Vincentelli_2004_Protein.Sci_13_2782
PubMedID: 15388864

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 : Medium-scale structural genomics: strategies for protein expression and crystallization - Vincentelli_2003_Acc.Chem.Res_36_165
Author(s) : Vincentelli R , Bignon C , Gruez A , Canaan S , Sulzenbacher G , Tegoni M , Campanacci V , Cambillau C
Ref : Acc Chem Res , 36 :165 , 2003
Abstract : While high-throughput methods of protein production and crystallization are beginning to be well documented, owing to the output of large structural genomics programs, medium-throughput methods at the laboratory scale lag behind. In this paper, we report a possible way for an academic laboratory to adapt high-throughput to medium-throughput methods, on the basis of the first results of two projects aimed at solving the 3D structures of Escherichia coli and Mycobacterium tuberculosis (Tb) proteins of unknown function. We have developed sequential and iterative procedures as well as new technical processes for these programs. Our results clearly demonstrate the value of this medium-throughput approach. For instance, in the first 14 months of the E. coli program, 69 out of 108 target genes led to soluble proteins, 36 were brought to crystallization, and 28 yielded crystals; among the latter, 13 led to usable data sets and 9 to structures. These results, still incomplete, might help in planning future directions of expression and crystallization of proteins applied to medium-throughput structural genomics programs.
ESTHER : Vincentelli_2003_Acc.Chem.Res_36_165
PubMedSearch : Vincentelli_2003_Acc.Chem.Res_36_165
PubMedID: 12641473

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 : Structure-activity of cutinase, a small lipolytic enzyme - Longhi_1999_Biochim.Biophys.Acta_1441_185
Author(s) : Longhi S , Cambillau C
Ref : Biochimica & Biophysica Acta , 1441 :185 , 1999
Abstract : Cutinase, a small lipolytic enzyme, is the smallest member of the alpha/beta-hydrolase fold family, to which the other lipases belong. Cutinase has a catalytic activity comparable to that of pancreatic lipase on short chain triglycerides, and retains a significant activity on long chain triglycerides. Cutinase has been extensively studied using site-directed mutagenesis, and we have thoroughly characterized it from a structural point of view. Besides the native enzyme, tens of mutants and several inhibitor complexes have been solved, providing a complete and precise picture of the structure, dynamics and catalytic machinery of cutinase.
ESTHER : Longhi_1999_Biochim.Biophys.Acta_1441_185
PubMedSearch : Longhi_1999_Biochim.Biophys.Acta_1441_185
PubMedID: 10570246

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 : Colipase: structure and interaction with pancreatic lipase - van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
Author(s) : van Tilbeurgh H , Bezzine S , Cambillau C , Verger R , Carriere F
Ref : Biochimica & Biophysica Acta , 1441 :173 , 1999
Abstract : Colipase is a small protein cofactor needed by pancreatic lipase for the efficient dietary lipid hydrolysis. It binds to the C-terminal, non-catalytic domain of lipase, thereby stabilising an active conformation and considerably increasing the overall hydrophobic binding site. Structural studies of the complex and of colipase alone have clearly revealed the functionality of its architecture. Interestingly, a structural analogy has recently been discovered between colipase and a domain in a developmental protein (Dickkopf), based on sequence analogy and homology modeling. Whether this structural analogy implies a common function (lipid interaction) remains to be clarified. Structural analogies have also been recognised between the pancreatic lipase C-terminal domain, the N-terminal domains of lipoxygenases and the C-terminal domain of alpha-toxin. These non-catalytic domains in the latter enzymes are important for interaction with membranes. It has not been established if these domains are also involved in eventual protein cofactor binding as is the case for pancreatic lipase.
ESTHER : van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
PubMedSearch : van Tilbeurgh_1999_Biochim.Biophys.Acta_1441_173
PubMedID: 10570245

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 : Packing forces in nine crystal forms of cutinase - Jelsch_1998_Proteins_31_320
Author(s) : Jelsch C , Longhi S , Cambillau C
Ref : Proteins , 31 :320 , 1998
Abstract : During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200-600 A2 range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts.
ESTHER : Jelsch_1998_Proteins_31_320
PubMedSearch : Jelsch_1998_Proteins_31_320
PubMedID: 9593202

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 : Pancreatic lipase structure-function relationships by domain exchange - Carriere_1997_Biochemistry_36_239
Author(s) : Carriere F , Thirstrup K , Hjorth S , Ferrato F , Nielsen PF , Withers-Martinez C , Cambillau C , Boel E , Thim L , Verger R
Ref : Biochemistry , 36 :239 , 1997
Abstract : We designed chimeric mutants by exchanging the lid domains of the classical human pancreatic lipase (HPL) and the guinea pig pancreatic lipase related protein 2 (GPLRP2). This latter enzyme possesses naturally a large deletion within the lid domain and is not activated by lipid/water interfaces. Furthermore, GPLRP2 exhibits phospholipase A1 and lipase activities in the same order of magnitude, whereas HPL has no significant phospholipase activity and displays a clear interfacial activation. An HPL mutant [HPL(-lid)] with GPLRP2 mini-lid domain does not display interfacial activation. Its specific activity toward triglycerides is, however, dramatically reduced. A GPLRP2 mutant [GPLRP2(+lid)] with HPL full-length lid domain is not interfacially activated, and its lid domain probably exists under a permanent open conformation. Therefore, the phenomenon of interfacial activation in HPL is not only due to the presence of a full-length lid domain but also to other structural elements which probably allow the existence of stabilized closed and open conformations of the lid. GPLRP2(+lid) phospholipase activity is significantly reduced as compared to GPLRP2, whereas its lipase activity remains at the same level. Therefore, the lid domain plays a major role in substrate selectivity and can be considered as part of the active site. However, the presence of a full-length lid domain is not sufficient to explain the absence of phospholipase activity in HPL since HPL(-lid) does not display any phospholipase activity. We also produced a chimeric GPLRP2 mutant in which the C-terminal domain was substituted by the HPL C-terminal domain. The colipase effects, i.e., anchoring and stabilization of the lipase at the interface, are clearly observed with the chimera, whereas GPLRP2 is insensitive to colipase. The kinetic characterization of this chimera reveals for the first time that the interfacial stability of pancreatic lipases depends on the structure of the C-terminal domain.
ESTHER : Carriere_1997_Biochemistry_36_239
PubMedSearch : Carriere_1997_Biochemistry_36_239
PubMedID: 8993339

Title : Crystal structure of cutinase covalently inhibited by a triglyceride analogue - Longhi_1997_Protein.Sci_6_275
Author(s) : Longhi S , Mannesse M , Verheij HM , De Haas GH , Egmond M , Knoops-Mouthuy E , Cambillau C
Ref : Protein Science , 6 :275 , 1997
Abstract : Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors which bear little resemblance to a natural triglyceride substrate In this article we describe the crystal structure of cutinase covalently inhibited by R)-1,2-dibutyl carbamoylglycero-3-O-p-nitrophenylbutyl-phos phonate a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway The structure which has been solved at 2.3 A reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice The overall shape of the inhibitor is that of a fork the two dibutyl carbamoyl chains point towards the surface of the protein whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn-1 and sn-2 chains The sn-3 chain is accommodated in a rather small pocket at the bottom of the active site crevice thus providing a structural explanation for the preference of cutinase for short acyl chain substrates
ESTHER : Longhi_1997_Protein.Sci_6_275
PubMedSearch : Longhi_1997_Protein.Sci_6_275
PubMedID: 9041628
Gene_locus related to this paper: fusso-cutas

Title : Pancreatic lipases and their complexes with colipases and inhibitors: crystallization and crystal packing -
Author(s) : Cambillau C , Bourne Y , Egloff MP , Martinez C , van Tilbeurgh H
Ref : Methods Enzymol , 284 :107 , 1997
PubMedID: 9379929

Title : Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis - Longhi_1997_J.Mol.Biol_268_779
Author(s) : Longhi S , Czjzek M , Lamzin V , Nicolas A , Cambillau C
Ref : Journal of Molecular Biology , 268 :779 , 1997
Abstract : X-ray data have been recorded to 1.0 A resolution from a crystal of Fusarium solani cutinase using synchrotron radiation and an imaging-plate scanner. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a considerable quality improvement in the electron density maps with respect to an intermediate isotropic model. The final model has an R-factor of 9.4%, with a mean coordinate error of 0.021 A, as estimated from inversion of the least-squares matrix. The availability of an accurate structure at atomic resolution and of meaningful estimates of the errors in its atomic parameters, allowed an extensive analysis of several stereochemical parameters, such as peptide planarity, main-chain and some side-chain bond distances. The hydrogen atoms could be clearly identified in the electron density, thus providing unambiguous evidence on the protonation state of the catalytic histidine residue. The atomic resolution revealed an appreciable extent of flexibility in the cutinase active site, which might be correlated with a possible adaptation to different substrates. The anisotropic treatment of thermal factors provided insights into the anisotropic nature of motions. The analysis of these motions in the two loops delimiting the catalytic crevice pointed out a "breath-like" movement in the substrate binding region of cutinase.
ESTHER : Longhi_1997_J.Mol.Biol_268_779
PubMedSearch : Longhi_1997_J.Mol.Biol_268_779
PubMedID: 9175860
Gene_locus related to this paper: fusso-cutas , orysa-LPL1

Title : A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig - Withers-Martinez_1996_Structure_4_1363
Author(s) : Withers-Martinez C , Carriere F , Verger R , Bourgeois D , Cambillau C
Ref : Structure , 4 :1363 , 1996
Abstract : BACKGROUND: The guinea pig pancreatic lipase-related protein 2 (GPLRP2) differs from classical pancreatic lipases in that it displays both lipase and phospholipase A1 activities; classical pancreatic lipases have no phospholipase activity. The sequence of GPLRP2 is 63 % identical to that of human pancreatic lipase (HPL), but the so-called lid domain, is much reduced in GPLRP2. A phospholipase A1 from hornet venom (Dolml PLA1) is very similar to HPL and GPLRP2 but is devoid of lipase activity; Dolml PLA1 also contains a reduced lid domain and lacks a region termed the beta9 loop, which is located in the vicinity of the HPL and GPLRP2 active sites. The structure determination of a chimera of GPLRP2 and HPL and domain building of Dolml PLA1 were undertaken to gain a better understanding of the structural parameters responsible for the differences in lipase versus phospholipase activity among these structurally related enzymes. RESULTS: The crystal structure of a chimeric mutant of GPLRP2, consisting of the catalytic domain of GPLRP2 and the C-terminal domain of HPL, has been solved and refined to 2.1 A resolution. This enzyme belongs to the alpha/beta hydrolase fold family and shows high structural homology with classical pancreatic lipases. The active site is closely related to those of serine esterases, except for an unusual geometry of the catalytic triad. Due to the reduced size of the lid domain, the catalytic serine is fully accessible to solvent. Part of the beta9 loop, which stabilizes the lid domain in the closed conformation of the classical HPL, is totally exposed to the solvent and is not visible in the electron-density map. CONCLUSIONS: The structures of the related enzymes, GPLRP2 and HPL and the model of Dolml PLA1, provide insights into the role played by the loops located above the active site in controlling substrate selectivity towards triglycerides or phospholipids. In GPLRP2, the lid domain is reduced in size compared to HPL, and hydrophilic residues are exposed to solvent. GPLRP2 is thus able to accommodate the polar head of phospholipids. The beta9 loop is still present in GPLRP2, making it possible for this enzyme to still accommodate triglycerides. In Dolml PLA1, the beta9 loop is absent, and this enzyme is unable to process triglycerides retaining only the phospholipase A1 activity.
ESTHER : Withers-Martinez_1996_Structure_4_1363
PubMedSearch : Withers-Martinez_1996_Structure_4_1363
PubMedID: 8939760
Gene_locus related to this paper: human-PNLIP

Title : Acyl glycerol hydrolases: inhibitors, interface and catalysis - Cambillau_1996_Curr.Opin.Struct.Biol_6_449
Author(s) : Cambillau C , Longhi S , Nicolas A , Martinez C
Ref : Current Opinion in Structural Biology , 6 :449 , 1996
Abstract : The last five years have witnessed the solution of a large number of lipase structures, which has led, among other insights, to the structural interpretation of the interfacial activation phenomenon in terms of 'lid' opening. This interpretation has been extended this year to include phospholipase A2. Recent structural studies on lipases have provided data on the detailed mechanisms underlying the behaviour of lipases: how they bind to inhibitors or substrates, and what interactions occur between their hydrophobic face and hydrophobic molecules, for example. In addition, studies on cutinase point mutants have shed some light on the role of the oxyanion hole in lipolytic catalysis.
ESTHER : Cambillau_1996_Curr.Opin.Struct.Biol_6_449
PubMedSearch : Cambillau_1996_Curr.Opin.Struct.Biol_6_449
PubMedID: 8794161

Title : Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants - Longhi_1996_Proteins_26_442
Author(s) : Longhi S , Nicolas A , Creveld L , Egmond M , Verrips CT , de Vlieg J , Martinez C , Cambillau C
Ref : Proteins , 26 :442 , 1996
Abstract : In characterizing mutants and covalently inhibited complexes of Fusarium solani cutinase, which is a 197-residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Taking advantage of this considerable body of information, a structural comparative analysis was carried out to investigate the dynamics of cutinase. Surface loops were identified as the major flexible protein regions, particularly those forming the active-site groove, whereas the elements constituting the protein scaffold were found to retain the same conformation in all the cutinase variants studied. Flexibility turned out to be correlated with thermal motion. With a given crystal packing environment, a high flexibility turned out to be correlated with a low involvement in crystal packing contacts. The high degree of crystal polymorphism, which allowed different conformations with similar energy to be detected, made it possible to identify motions which would have remained unidentified if only a single crystal form had been available. Fairly good agreement was found to exist between the data obtained from the structural comparison and those from a molecular dynamics (MD) simulation carried out on the native enzyme. The crystallographic approach used in this study turned out to be a suitable tool for investigating cutinase dynamics. Because of the availability of a set of closely related proteins in different crystal environments, the intrinsic drawback of a crystallographic approach was bypassed. By combining several static pictures, the dynamics of the protein could be monitored much more realistically than what can be achieved on the basis of static pictures alone.
ESTHER : Longhi_1996_Proteins_26_442
PubMedSearch : Longhi_1996_Proteins_26_442
PubMedID: 8990497
Gene_locus related to this paper: fusso-cutas

Title : Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state - Nicolas_1996_Biochemistry_35_398
Author(s) : Nicolas A , Egmond M , Verrips CT , de Vlieg J , Longhi S , Cambillau C , Martinez C
Ref : Biochemistry , 35 :398 , 1996
Abstract : Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.
ESTHER : Nicolas_1996_Biochemistry_35_398
PubMedSearch : Nicolas_1996_Biochemistry_35_398
PubMedID: 8555209
Gene_locus related to this paper: fusso-cutas

Title : The 2.46 A resolution structure of the pancreatic lipase-colipase complex inhibited by a C11 alkyl phosphonate - Egloff_1995_Biochemistry_34_2751
Author(s) : Egloff MP , Marguet F , Buono G , Verger R , Cambillau C , van Tilbeurgh H
Ref : Biochemistry , 34 :2751 , 1995
Abstract : Pancreatic lipase belongs to the serine esterase family and can therefore be inhibited by classical serine reagents such as diisopropyl fluoride or E600. In an attempt to further characterize the active site and catalytic mechanism, we synthesized a C11 alkyl phosphonate compound. This compound is an effective inhibitor of pancreatic lipase. The crystal structure of the pancreatic lipase-colipase complex inhibited by this compound was determined at a resolution of 2.46 A and refined to a final R-factor of 18.3%. As was observed in the case of the structure of the ternary pancreatic lipase-colipase-phospholipid complex, the binding of the ligand induces rearrangements of two surface loops in comparison with the closed structure of the enzyme (van Tilbeurgh et al., 1993b). The inhibitor, which could be clearly observed in the active site, was covalently bound to the active site serine Ser152. A racemic mixture of the inhibitor was used in the crystallization, and there exists evidence that both enantiomers are bound at the active site. The C11 alkyl chain of the first enantiomer fits into a hydrophobic groove and is though to thus mimic the interaction between the leaving fatty acid of a triglyceride substrate and the protein. The alkyl chain of the second enantiomer also has an elongated conformation and interacts with hydrophobic patches on the surface of the open amphipathic lid. This may indicate the location of a second alkyl chain of a triglyceride substrate. Some of the detergent molecules, needed for the crystallization, were also observed in the crystal. Some of them were located at the entrance of the active site, bound to the hydrophobic part of the lid. On the basis of this crystallographic study, a hypothesis about the binding mode of real substrates and the organization of the active site is proposed.
ESTHER : Egloff_1995_Biochemistry_34_2751
PubMedSearch : Egloff_1995_Biochemistry_34_2751
PubMedID: 7893686
Gene_locus related to this paper: human-PNLIP

Title : Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase - Egloff_1995_Protein.Sci_4_44
Author(s) : Egloff MP , Sarda L , Verger R , Cambillau C , van Tilbeurgh H
Ref : Protein Science , 4 :44 , 1995
Abstract : Colipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3-A-resolution X-ray structures of lipase-colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C-terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase-colipase complex. From the 2.4-A-resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206-211). Colipase lacks well-defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger-like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C-terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.
ESTHER : Egloff_1995_Protein.Sci_4_44
PubMedSearch : Egloff_1995_Protein.Sci_4_44
PubMedID: 7773176

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

Title : Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution - Bourne_1994_J.Mol.Biol_238_709
Author(s) : Bourne Y , Martinez C , Kerfelec B , Lombardo D , Chapus C , Cambillau C
Ref : Journal of Molecular Biology , 238 :709 , 1994
Abstract : Pancreatic lipase (EC 3.1.1.3) plays a key role in dietary fat digestion by converting triacylglycerols into 2-monoacylglycerols and free fatty acids in the intestine. Although the crystallographic structures of the human pancreatic lipase and of a human lipase-porcine colipase complex have been solved, no refined structure of pancreatic lipase has yet been published. The crystal structure of the horse enzyme was solved by the molecular replacement method from the model of the human pancreatic lipase and subsequently refined to 2.3 A resolution. The final model contains two molecules of 449 amino acid residues each in the asymmetric unit, 705 well-defined water molecules and two calcium ions. The two molecules in the asymmetric unit of the orthorhombic crystals are related by a 2-fold non-crystallographic symmetry axis as in the case of the human lipase. However, the association between the two molecules in their respective crystal forms is different. The overall molecular structure of the horse lipase is very similar to that of the human enzyme. The horse lipase is made up of two well-defined domains. The N-terminal domain which bears the active centre has a typical alpha/beta hydrolase fold topology. The C-terminal domain which is devoted to colipase binding has a beta-sheet sandwich topology. Comparison of equivalent C alpha atom positions between the final model of the horse lipase and the human lipase structure shows only slight differences which are mainly located in the C-terminal domain. The horse enzyme possesses the common features of the known mammalian and microbial lipases, in particular the "flap" covering the catalytic triad. In addition to more precise information concerning these features, the elucidation of the horse lipase crystal structure allowed us to better understand the structural basis of the kinetic behaviour of pancreatic lipases towards a soluble substrate, p-nitrophenyl acetate, and the different sensitivity of these enzymes towards limited proteolysis.
ESTHER : Bourne_1994_J.Mol.Biol_238_709
PubMedSearch : Bourne_1994_J.Mol.Biol_238_709
PubMedID: 8182745
Gene_locus related to this paper: horse-1plip

Title : Cutinase, a lipolytic enzyme with a preformed oxyanion hole - Martinez_1994_Biochemistry_33_83
Author(s) : Martinez C , Nicolas A , van Tilbeurgh H , Egloff MP , Cudrey C , Verger R , Cambillau C
Ref : Biochemistry , 33 :83 , 1994
Abstract : Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.
ESTHER : Martinez_1994_Biochemistry_33_83
PubMedSearch : Martinez_1994_Biochemistry_33_83
PubMedID: 8286366
Gene_locus related to this paper: fusso-cutas , orysa-LPL1

Title : Engineering cysteine mutants to obtain crystallographic phases with a cutinase from Fusarium solani pisi - Martinez_1993_Protein.Eng_6_157
Author(s) : Martinez C , de Geus P , Stanssens P , Lauwereys M , Cambillau C
Ref : Protein Engineering , 6 :157 , 1993
Abstract : Cutinases are extracellular enzymes involved in the disruption of cutine, an insoluble polyester which covers the surface of plants. They belong to a class of serine esterases that are able to hydrolyse fatty acid esters and emulsified triglycerides as efficiently as lipases, but without displaying interfacial activation. Classical crystallographic methods for obtaining heavy-atom derivatives failed, so the cutinase structure has been solved exclusively by the multiple isomorphous replacement method using four Hg derivatives obtained from mutants S4C, S92C, S120C and S129C. Two of these derivatives behaved as expected: (i) the cys mutant of the catalytic Ser S120C, located at the surface of the active site pocket, leads to a good derivative; and (ii) the Hg atom of the derivative obtained with the S92C mutant is completely accessible to the solvent and occupies two alternative positions--consequently a poor derivative results. In contrast, two mutants show an unexpected behaviour: (i) the Hg atom in the S129C mutant was completely buried 10 A below the protein surface and yielded the best derivative; and (ii) a poor quality derivative was obtained with the S4C mutant. Cys 4 belongs to the disordered propeptide 1-16. The Cys 4 bound Hg atom is located in front of the Asp58 side chain, but neither Cys4 nor parts of the propeptide are clearly visible in the electron density maps of the derivative structure.
ESTHER : Martinez_1993_Protein.Eng_6_157
PubMedSearch : Martinez_1993_Protein.Eng_6_157
PubMedID: 8475042
Gene_locus related to this paper: fusso-cutas

Title : Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography - van Tilbeurgh_1993_Nature_362_814
Author(s) : van Tilbeurgh H , Egloff MP , Martinez C , Rugani N , Verger R , Cambillau C
Ref : Nature , 362 :814 , 1993
Abstract : The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).
ESTHER : van Tilbeurgh_1993_Nature_362_814
PubMedSearch : van Tilbeurgh_1993_Nature_362_814
PubMedID: 8479519
Gene_locus related to this paper: human-PNLIP

Title : A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase - Hjorth_1993_Biochemistry_32_4702
Author(s) : Hjorth A , Carriere F , Cudrey C , Woldike H , Boel E , Lawson DM , Ferrato F , Cambillau C , Dodson GG , Thim L , et al.
Ref : Biochemistry , 32 :4702 , 1993
Abstract : Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.
ESTHER : Hjorth_1993_Biochemistry_32_4702
PubMedSearch : Hjorth_1993_Biochemistry_32_4702
PubMedID: 8490016
Gene_locus related to this paper: cavpo-2plrp , ratno-1plip

Title : Isoform purification of gastric lipases. Towards crystallization - Moreau_1992_J.Mol.Biol_225_147
Author(s) : Moreau H , Abergel C , Carriere F , Ferrato F , Fontecilla-Camps JC , Cambillau C , Verger R
Ref : Journal of Molecular Biology , 225 :147 , 1992
Abstract : Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.
ESTHER : Moreau_1992_J.Mol.Biol_225_147
PubMedSearch : Moreau_1992_J.Mol.Biol_225_147
PubMedID: 1583687

Title : Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent - Martinez_1992_Nature_356_615
Author(s) : Martinez C , de Geus P , Lauwereys M , Matthyssens G , Cambillau C
Ref : Nature , 356 :615 , 1992
Abstract : Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.
ESTHER : Martinez_1992_Nature_356_615
PubMedSearch : Martinez_1992_Nature_356_615
PubMedID: 1560844
Gene_locus related to this paper: fusso-cutas , orysa-LPL1

Title : Structure of the pancreatic lipase-procolipase complex - van Tilbeurgh_1992_Nature_359_159
Author(s) : van Tilbeurgh H , Sarda L , Verger R , Cambillau C
Ref : Nature , 359 :159 , 1992
Abstract : Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.
ESTHER : van Tilbeurgh_1992_Nature_359_159
PubMedSearch : van Tilbeurgh_1992_Nature_359_159
PubMedID: 1522902
Gene_locus related to this paper: human-PNLIP

Title : Effects of gene mutations in lipoprotein and hepatic lipases as interpreted by a molecular model of the pancreatic triglyceride lipase - Derewenda_1991_J.Biol.Chem_266_23112
Author(s) : Derewenda ZS , Cambillau C
Ref : Journal of Biological Chemistry , 266 :23112 , 1991
Abstract : A molecular model of human pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. (1990) Nature 343, 771-774) is used to explain the possible structural effects of the amino acid mutations identified to date in the human lipoprotein and hepatic lipase genes. A sequence homology profile was used to evaluate the alignment of the amino acid sequences of all three lipolytic enzymes (Kirchgessner, T. G., Chuat, J.-C., Heinzmann, C., Etienne, J., Guilhot, S., Svenson, K., Ameis, D., Pilon, C., D'Auriol, L., Andalibi, A., Schotz, M. C., Galibert, F., and Lusis, A. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9647-9651) with respect to the secondary structure elements identified in the pancreatic lipase. As expected, maximum homology is observed in internal regions namely the hydrophobic strands of the central beta-pleated sheet. This observation strongly supports the hypothesis that all three molecules exhibit a very similar three-dimensional structure, particularly in the N-terminal catalytic domain. There is considerable variation in some of the surface loops connecting the individual strands, whereas others are conserved. It is hypothesized that the most conserved loops located around the active site are responsible for the catalytic function (similar for all three enzymes), whereas those that markedly differ are involved in the regulation at the molecular level, namely the binding of colipase (pancreatic enzyme) and apolipoprotein CII (lipoprotein lipase). The currently available library of hepatic and lipoprotein gene mutations seems to indicate that the majority of mutants disrupt the folding of the polypeptide chain, rather than affect specific constellations in and around the catalytic site or regulatory loops.
ESTHER : Derewenda_1991_J.Biol.Chem_266_23112
PubMedSearch : Derewenda_1991_J.Biol.Chem_266_23112
PubMedID: 1744109

Title : Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi - Abergel_1990_J.Mol.Biol_215_215
Author(s) : Abergel C , Martinez C , Fontecilla-Camps JC , Cambillau C , de Geus P , Lauwereys M
Ref : Journal of Molecular Biology , 215 :215 , 1990
Abstract : Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).
ESTHER : Abergel_1990_J.Mol.Biol_215_215
PubMedSearch : Abergel_1990_J.Mol.Biol_215_215
PubMedID: 2213880

Title : Crystallization and preliminary X-ray study of horse pancreatic lipase - Lombardo_1989_J.Mol.Biol_205_259
Author(s) : Lombardo D , Chapus C , Bourne Y , Cambillau C
Ref : Journal of Molecular Biology , 205 :259 , 1989
Abstract : Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.
ESTHER : Lombardo_1989_J.Mol.Biol_205_259
PubMedSearch : Lombardo_1989_J.Mol.Biol_205_259
PubMedID: 2926806
Gene_locus related to this paper: horse-1plip