Crystal Structure of Human Gastric Lipase and Model of Lysosomal Acid Lipase, Two Lipolytic Enzymes of Medical Interest

Artigo Acesso aberto Revisado por pares

Crystal Structure of Human Gastric Lipase and Model of Lysosomal Acid Lipase, Two Lipolytic Enzymes of Medical Interest

1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês

10.1074/jbc.274.24.16995

ISSN

1083-351X

Autores

Alain Roussel, Stéphane Canaan, Marie-Pierre Egloff, Mireille Rivière, Liliane Dupuis, Robert Verger, Christian Cambillau,

Tópico(s)

Lysosomal Storage Disorders Research

Resumo

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-Å 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 α/β 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. 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-Å 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 α/β 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. Since 1990, when the first three-dimensional structures of a fungal (Rhizomucor miehei lipase) and a mammalian lipase (human pancreatic lipase (HPL)) 1The abbreviations used are: HPL, human pancreatic lipase; HGL, human gastric lipase; rHGL, recombinant HGL; HLAL, human lysosomal acid lipase; SIRAS, single isomorphous replacement with anomalous scattering; HPP, human protective protein 1The abbreviations used are: HPL, human pancreatic lipase; HGL, human gastric lipase; rHGL, recombinant HGL; HLAL, human lysosomal acid lipase; SIRAS, single isomorphous replacement with anomalous scattering; HPP, human protective protein were published, growing interest in lipolysis has led to the structural determination of several lipases of various origins, including those present in bacteria, fungi, and mammals. All the lipases investigated so far vary considerably in size and in their amino acid sequences. However, they are all serine esterases belonging to the α/β hydrolase superfamily (1Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1835) Google Scholar) in which the nucleophilic serine, part of a Ser-His-(Asp/Glu) triad, is located in an extremely sharp turn (nucleophilic elbow). Another feature that is common to all the members of the α/β hydrolase superfamily as well as to proteases is the occurrence of an oxyanion hole, which stabilizes the transition state. Some organophosphorous compounds inhibit lipases in a similar way to what occurs in the case of serine proteases (2Kraut J. Annu. Rev. Biochem. 1977; 46: 331-358Crossref PubMed Scopus (1073) Google Scholar). The three-dimensional structures of several complexes consisting of lipases bound to covalent inhibitors have been solved: R. miehei lipase (3Derewenda Z.S. Derewenda U. Dodson G.G. J. Mol. Biol. 1992; 227: 818-839Crossref PubMed Scopus (250) Google Scholar, 4Brzozowski A.M. Derewenda U. Derewenda Z.S. Dodson G.G. Lawson D.M. Turkenburg J.P. Bjorkling F. Huge-Jensen B. Patkar S.A. Thim L. Nature. 1991; 351: 491-494Crossref PubMed Scopus (1047) Google Scholar) and Candida antartica B lipases bound to a C6-alkyl phosphonate (5Uppenberg J. Ohrner N. Norin M. Hult K. Kleywegt G.J. Patkar S. Waagen V. Anthonsen T. Jones T.A. Biochemistry. 1995; 34: 16838-16851Crossref PubMed Scopus (450) Google Scholar), Candida rugosa lipase bound to long chain alkyl sulfonyl (6Grochulski P. Bouthillier F. Kazlauskas R.J. Serreqi A.N. Schrag J.D. Ziomek E. Cygler M. Biochemistry. 1994; 33: 3494-3500Crossref PubMed Scopus (240) Google Scholar), Pseudomonas cepacialipase (7Lang D. Mannesse M. de Haas G. Verheij H. Dijkstra B. Eur. J. Biochem. 1998; 254: 333-340Crossref PubMed Scopus (142) Google Scholar) as well as cutinase (8Longhi S. Mannesse M. Verheij H.M. de Haas G.H. Egmond M. Knoops-Mouthuy E. Cambillau C. Protein Sci. 1997; 6: 275-286Crossref PubMed Scopus (86) Google Scholar) bound to a dialkylcarbamoylglycerophosphonate, and human pancreatic lipase-colipase complex bound to C11-alkyl phosphonate (9Egloff M.-P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (258) Google Scholar). It has been established that the covalently inhibited lipases are in the so called “open” conformation, i.e. that the lid has moved away to give free access to the active-site serine. It has been suggested that this mechanism may be instrumental in the binding of lipases to the water-lipid interface and that the presence of a lid in the structure of the enzyme may be involved in the interfacial activation process (4Brzozowski A.M. Derewenda U. Derewenda Z.S. Dodson G.G. Lawson D.M. Turkenburg J.P. Bjorkling F. Huge-Jensen B. Patkar S.A. Thim L. Nature. 1991; 351: 491-494Crossref PubMed Scopus (1047) Google Scholar,10van Tilbeurgh H. Egloff M.-P. Martinez C. Rugani N. Verger R. Cambillau C. Nature. 1993; 362: 814-820Crossref PubMed Scopus (633) Google Scholar). Among the mammalian lipases, the acid lipases belong to a family of enzymes that have the ability to withstand acidic conditions. This family that includes the preduodenal lipases and human lysosomal lipase shows no sequence homology with any other known lipase families (11Petersen S.B. Drabløs F. Woolley P. Petersen S. Lipases: Their Biochemistry, Structure, and Application. Cambridge University Press, Cambridge, England1994: 23-48Google Scholar). The preduodenal lipases form a group of closely related enzymes originating either from the stomach, the tongue, or the pharynx (12Carrière F. Gargouri Y. Moreau H. Ransac S. Rogalska E. Verger R. Woolley P. Petersen S.B. Lipases: Their Structure, Biochemistry, and Application. Cambridge University Press, Cambridge, England1994: 181-205Google Scholar). They all have a low pH optimum, and none of them require any specific protein cofactor. Human gastric lipase (HGL, EC 3.1.1.3) is secreted by the chief cells located in the fundic part of the stomach (13Moreau H. Bernadac A. Gargouri Y. Benkouka F. Laugier R. Verger R. Histochemistry. 1989; 91: 419-423Crossref PubMed Scopus (61) Google Scholar), where it initiates the digestion of triacylglycerols (14Carrière F. Barrowman J.A. Verger R. Laugier R. Gastroenterology. 1993; 105: 876-888Abstract Full Text PDF PubMed Google Scholar, 15Hamosh M. Hamosh M. Lingual and Gastric Lipases: Their Role in Fat Digestion. CRC Press, Inc., Boston1990Google Scholar). The maximum specific activities of HGL are 1160 units/mg on TC4 (pH 6.0), 1110 units/mg on TC8 (pH 6.0), and 600 units/mg on IntralipidTM (pH 5.0) (16Gargouri Y. Piéroni G. Rivière C. Saunière J.-F. Lowe P.A. Sarda L. Verger R. Gastroenterology. 1986; 91: 919-925Abstract Full Text PDF PubMed Google Scholar). Native HGL has an apparent molecular mass of 50 kDa and is a highly glycosylated molecule with 4 potentialN-glycosylation sites (17Bodmer M.W. Angal S. Yarranton G.T. Harris T.J.R. Lyons A. King D.J. Piéroni G. Rivière C. Verger R. Lowe P.A. Biochim. Biophys. Acta. 1987; 909: 237-244Crossref PubMed Scopus (138) Google Scholar). The glycan moiety was estimated to account for around 15% of its total protein mass (18Moreau H. Abergel C. Carrière F. Ferrato F. Fontecilla-Camps J.C. Cambillau C. Verger R. J. Mol. Biol. 1992; 225: 147-153Crossref PubMed Scopus (37) Google Scholar). This enzyme plays a crucial role in newborns, because pancreatic lipase is not yet fully developed at this age (15Hamosh M. Hamosh M. Lingual and Gastric Lipases: Their Role in Fat Digestion. CRC Press, Inc., Boston1990Google Scholar). The physiological importance of gastric lipase has been suspected for some time, based on pathological situations involving pancreatic exocrine insufficiency, such as the late stage of chronic pancreatitis or cystic fibrosis. In these cases, even in the complete absence of pancreatic lipase, the patients still absorb a high percentage of their ingested dietary fat (19Muller D.P.R. McCollum J.P.K. Tompeter R.S. Harries J.T. Gut. 1975; 16: 838PubMed Google Scholar, 20Lapey A. Kattwinkel J. di Sant Agnese P.A. Laster L. J. Pediatr. 1974; 84: 328-334Abstract Full Text PDF PubMed Scopus (85) Google Scholar). In substitutive enzymatic therapy, the use of acidic-resistant lipases should in principle yield more satisfactory results than the pancreatic preparations currently in use. The co-administration of acidic lipases, which hydrolyze dietary lipids under acidic conditions, should help to treat patients with various forms of pancreatic deficiency. Physiological studies have shown that preduodenal lipases are capable of acting not only in the stomach but also in the duodenum in synergy with a pancreatic lipase (14Carrière F. Barrowman J.A. Verger R. Laugier R. Gastroenterology. 1993; 105: 876-888Abstract Full Text PDF PubMed Google Scholar). Various clinical studies have been conducted on both animals and humans to assess the efficacy of enzymatic replacement therapies using acid-resistant lipases to treat exocrine pancreatic insufficiency (21Suzuki A. Mizumoto A. Sarr M.G. Dimagno E.P. Gastroenterology. 1997; 112: 2048-2055Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This treatment significantly increased the weight and reduced the steatorrhea in dogs. Despite the close amino acid sequence similarities (59% of the amino acids are identical) between HGL (17Bodmer M.W. Angal S. Yarranton G.T. Harris T.J.R. Lyons A. King D.J. Piéroni G. Rivière C. Verger R. Lowe P.A. Biochim. Biophys. Acta. 1987; 909: 237-244Crossref PubMed Scopus (138) Google Scholar) and human lysosomal acid lipase (HLAL, EC 3.1.1.3) (22Ameis D. Merkel M. Eckerskorn C. Greten H. Eur. J. Biochem. 1994; 219: 905-914Crossref PubMed Scopus (79) Google Scholar, 23Anderson R.A. Sando G.N. J. Biol. Chem. 1991; 266: 22479-22484Abstract Full Text PDF PubMed Google Scholar), HGL lacks the cholesteryl ester hydrolase activity reported in HLAL. The latter enzyme hydrolyzes not only the triglycerides that are delivered to the lysosomes by low density lipoprotein receptor-mediated endocytosis but also cholesteryl esters (24Goldstein J.L. Brown M.S. Annu. Rev. Biochem. 1977; 46: 897-930Crossref PubMed Scopus (1610) Google Scholar). The cholesterol released by this reaction plays an important regulatory role in cellular sterol metabolism. Defective HLAL activity has been found to be associated with two rare autosomal recessive traits, Wolman disease and cholesteryl ester storage disease. In Wolman disease (25Patrick A.D. Lake B.D. Nature. 1969; 222: 1067-1068Crossref PubMed Scopus (135) Google Scholar), a lack of HLAL activity results in a pronounced accumulation of cholesteryl esters and triacylglycerols in the lysosomes in most of the body tissues. The patients usually succumb to hepatic and adrenal failure within the first year of life. Cholesteryl ester storage disease, the other clinically recognized phenotypic form of HLAL deficiency, follows a more benign clinical course (26Burke J. Schubert W. Science. 1972; 176: 309-310Crossref PubMed Scopus (66) Google Scholar), and a residual HLAL activity has been detected. Since the cloning of the cDNA and determination of the genomic organization of the gene (LIPA) located on chromosome 10, which encodes HLAL (22Ameis D. Merkel M. Eckerskorn C. Greten H. Eur. J. Biochem. 1994; 219: 905-914Crossref PubMed Scopus (79) Google Scholar, 23Anderson R.A. Sando G.N. J. Biol. Chem. 1991; 266: 22479-22484Abstract Full Text PDF PubMed Google Scholar,27Aslanidis C. Klima H. Lackner K.J. Schmitz G. Genomics. 1994; 20: 329-331Crossref PubMed Scopus (55) Google Scholar), some deleterious LIPA gene mutations have been identified (28Aslanidis C. Ries S. Fehringer P. Büchler C. Klima H. Schmitz G. Genomics. 1996; 33: 85-93Crossref PubMed Scopus (126) Google Scholar, 29Pagani F. Garcia R. Pariyarath R. Stuani C. Gridelli B. Paone G. Baralle F.E. Hum. Mol. Genet. 1996; 5: 1611-1617Crossref PubMed Scopus (43) Google Scholar, 30Pariyarath R. Pagani F. Stuani C. Garcia R. Baralle F.E. FEBS Lett. 1996; 397: 79-82Crossref PubMed Scopus (11) Google Scholar, 31Ameis D. Brockmann G. Knoblich R. Merkel M. Ostlund R.E. Yang J.W. Coates P.M. Cortner J.A. Feinman S.V. Greten H. J. Lipid Res. 1995; 36: 241-250Abstract Full Text PDF PubMed Google Scholar, 32Seedorf U. Wiebusch H. Muntoni S. Christensen N.C. Skovby F. Nickel V. Roskos M. Funke H. Ose L. Assmann G. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 773-778Crossref PubMed Scopus (40) Google Scholar, 33Anderson R.A. Byrum R.S. Coates P.M. Sando G.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2718-2722Crossref PubMed Scopus (103) Google Scholar, 34Pagani F. Zagato L. Merati G. Paone G. Gridelli B. Maier J.A. Hum. Mol. Genet. 1994; 3: 1605-1609Crossref PubMed Scopus (29) Google Scholar). Most of these mutations affect either the mRNA splicing or the amino acid sequence of HLAL. The exact correlations between these mutations and the biochemical and clinical phenotypes still remains to be elucidated, however. In this study, the crystal structure of recombinant HGL at 3.0-Å resolution is determined, and a model of HLAL is discussed and used to possibly explain the previously reported cholesteryl ester storage disease mutations. rHGL was expressed in the baculovirus/insect cell system (48Wicker-Planquart C. Canaan S. Rivière M. Dupuis L. Verger R. Protein Eng. 1996; 9: 1225-1232Crossref PubMed Scopus (28) Google Scholar). The active enzyme was produced on a large scale (5–13 mg/liter) from recombinant baculovirus-infected insect cells using a bioreactor and its specific activity (μmole·min−1·mg−1) was around 700 units/mg (49Canaan S. Dupuis L. Rivière M. Faessel K. Romette J.L. Verger R. Wicker-Planquart C. Protein Expression Purif. 1998; 14: 23-30Crossref PubMed Scopus (31) Google Scholar). The amino acid sequence (KLHPG) of rHGL in the purified protein starts at residue 4. Crystallization experiments were performed using the hanging-drop vapor diffusion method. Crystals of the rHGL were obtained by mixing 2 μl of a well solution (2 m ammonium sulfate, 1.4% tert-butanol, at pH 5.0) with 2 μl of a protein solution at 5–6 mg/ml. The crystals are cubic, space group I 21 3 with cell dimensions a = b = c = 244.0 Å3. The protein mass, 47,673 Da, was determined from collected crystals by matrix-assisted laser desorption ionization time-of-flight spectroscopy. There are two molecules in the asymmetric unit (see below), and the V m was estimated to be 6.35 Å3/Da (81% water content). For the mercury derivative preparation, the crystals were transferred in a synthetic liquor corresponding to the well solution containing 23 mmmercuric acetate. To assess the catalytic activity of the crystallized enzyme, tests were performed on both dissolved and intact crystals. Ten crystals were washed three times in the crystallization buffer and were subsequently dissolved in 50 μl of water, and the protein concentration was estimated by absorbance at 230 nm. The lipase activity was measured titrimetrically at 37 °C using a pH stat (metrohm) at pH 5.7 with a tributyrin emulsion as the substrate: 0.5 ml of tributyrin added to 14.5 ml of 150 mm NaCl, 2 mm taurodeoxycholate, and 2 mm bovine serum albumin (16Gargouri Y. Piéroni G. Rivière C. Saunière J.-F. Lowe P.A. Sarda L. Verger R. Gastroenterology. 1986; 91: 919-925Abstract Full Text PDF PubMed Google Scholar). The specific activity (μmole·min−1·mg−1) was calculated and found to be around 580 units/mg. To test the catalytic activityin situ, a single rHGL crystal was incubated in the crystallization solution containing 0.1 mm Nitroblue tetrazolium and 0.75 mm 5 bromo-chloro-3-indoyl butyrate as the substrate. After 24 h of incubation, the crystal was intensely colored in blue/gray. All data sets were collected at 100 K using a cryo-stream cooler from Oxford Cryosystems. A first native and a derivative data set were collected in-house using a 300-mm MAR Research imaging plate detector mounted on a RU200 rotating anode generator (Rigaku, Tokyo, Japan). The generator was operated at 3.2 kW with a focal spot size of 0.3 × 0.3 mm2. A second native data set was collected at LURE (Orsay, France) on DW32 beamline at 0.963 Å wavelength using a 345-mm MAR Research imaging plate. All data were collected in frames of 1.0 degree and processed with DENZO. The scaling was performed with SCALA (CCP4 (50SERC Daresbury Laboratory Collaborative Computing Project 4. A Suite of Programs for Protein Crystallography.Acta. Crystallogr. Sec. D. 1994; 50: 760Crossref PubMed Scopus (19728) Google Scholar)), and the derivative was merged with FHSCAL. Data collection statistics are given in Table I. The fact that most of the crystals diffracted only very poorly made the heavy metal derivative search laborious. In the end, it led to one mercury derivative diffracting to 3.6-Å resolution, isomorphous to the native crystal.Table IData collection, phasing and refinement statisticsNative 1Hg derivativeNative 2Resolution (Å)15–4.115–3.615–3.0Completeness (%)97.9 (99.4)98.9 (95.6)99.6 (100)R-merge (highest shell) (%)26.9 (47.5)16.7 (34.7)6.8 (39.9)I/ς I2.7 (1.6)3.6 (2.1)10.9 (2.0)Redundancy5.3 (5.2)3.1 (3.1)4.0 (4.1)Riso28.4 (15–4.1 Å)Rcullis0.85 acentric/ 0.76 centricFigure of merit0.268 (0.255 acentric/ 0.456 centric)Phasing power1.14 acentric/ 0.9 centricRefinement statisticsResolution15.0–3.0R / Rfree(%)22.5 / 25.1Nr reflections45522 (2381 free)Nr atoms6194ς bond (Å) / ς angle (°)0.007/ 1.392Ramachandran Procheck (61) zones 1–3 (%)85.5 / 13.3 / 1.2 Open table in a new tab The position of the heavy metal was determined using Patterson methods. The MLPHARE software program (51Otwinowski Z. Evans P. Leslie A. Isomorphous Replacement and Anomalous Scattering. SERC Daresbury Laboratory, Warrington WA4, UK1991: 80-85Google Scholar) was then used to refine the heavy atom parameters and calculate phases to 4.0 Å, taking into account both isomorphous and anomalous differences. Resulting single isomorphous replacement with anomalous scattering (SIRAS) phases were considerably improved after flattening 80% of the solvent using density modification. At this stage, the map indicated clearly that the handedness of the helices was wrong. Repeating the previous steps with the heavy atom position giving the negative coordinates yielded a suitable map for determining an envelope for each of the two molecules in the asymmetric unit. The noncrystallographic symmetry operators were determined using both the GLRF program and the relation between the two molecular replacement solutions (automated molecular replacement density modification (52Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar)) obtained from a search using a bones model (MAPMAN (53Kleywegt G. Jones T. From First Map to Final Model. EPSRC, Daresbury Warrington WA4, UK1994: 59-66Google Scholar)) calculated on the basis of one molecule. The density modification program was then used again to improve the initial phases (from MLPHARE) by performing simultaneously solvent flattening, histogram matching, 2-fold noncrystallographic symmetry averaging, and phase extension. The best map was obtained when a suitable mask around one of the molecules was added to the program. The resolution of the derivative data set was actually better than that of the native one, and the map was therefore calculated using the derivative structure factor amplitudes with the phases extended to 3.6 Å. The model was built with the program TURBO-FRODO (54Roussel A. Cambillau C. Graphics S. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1991: 86Google Scholar) using the recently developedab-initio building tools, which make it possible to build a model from planar pseudo-residues in a very short time. When the connectivity and the direction of the polypeptide chain have been determined, the pseudo-residues are automatically replaced by the actual residues in the sequence. Side-chain fitting is then performed manually. When most of the model had been built, a 3.0-Å native data set was collected on synchrotron radiation at the LURE. These better data were then used to calculate a new map, with which the model was completed (370 residues of 376 in the recombinant protein). The four sugar glycosylation sites were already clearly identified in this experimental electron density map. Refinement was carried out in X-PLOR (55Brünger A.T. X-PLOR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar) against the 3.0-Å data. Five percent of the data were set aside for calculating and monitoring of the free R-factor (Rfree). Refinement involved cycles of simulated annealing using the slow-cool procedure interspersed with manual rebuilding. Noncrystallographic symmetry restraints were applied during the whole refinement procedure. Individual B-factor refinement was subsequently performed. The final model consisted of 6194 atoms; the final R-factor was 22.5%, and the R-free factor was 25.1%. The complete refinement statistics, given in Table I, the quality of the Ramachandran plot, and the electron density indicate that the model is better than expected for a structure at 3.0-Å resolution. Maps calculated from the derivative structure factor amplitudes showed a clear electron density for the mercury derivative bound to cysteine 244 but no significant movements of any part of the model. Fig. 1 was drawn up with Molscript (56Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster 3D (57Merrit E.A. Murphy M.E.P. Acta Crystallogr. Sec. D. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar), Fig. 2 with Clustal W (58Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55386) Google Scholar) and Alscript (59Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar), Figs. 3, 4, and 6 with Turbo-Frodo (54Roussel A. Cambillau C. Graphics S. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1991: 86Google Scholar), and Fig. 5 with GRASP (60Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-286Crossref PubMed Scopus (5314) Google Scholar). The coordinates and structure factor amplitudes have been deposited in the Protein Data Bank with entry code 1HLG.Figure 2Sequence alignment of acid preduodenal lipases and human lysosomal acid lipase. Note that there are no insertions and only one deletion in the alignment. Identities are displayed with yellow shading, and the values with respect to HGL are: HLAL, 59%; rabbit gastric lipase, 85%; dog gastric lipase, 85%; rat lingual lipase, 76%; calf pregastric esterase, 75%. The catalytic triad, Ser-153, His-353, and Asp-324 is ingreen, blue, and red, respectively. The oxyanion hole residues are boxed. The secondary structures have been calculated with DSSP.View Large Image Figure ViewerDownload (PPT)Figure 3The active site. A, stereo view of the electron density map around the active site residues Ser-153, His-353, and the single cysteine 244 (The density has been contoured at 1 ς level). B, representation of the HGL catalytic triad and of the oxyanion hole (Gln-154, Leu-67) superposed to the same part of HPL (green, in Cα tracing).View Large Image Figure ViewerDownload (PPT)Figure 4Representations of human protective protein (A) and rHGL (B) in the same orientation with the catalytic triad in red. A, Corey-Pauling-Koltun view of the core domain of HPP (yellow) and of its cap domain (blue). The putative excision peptide is given by a Cα trace (green). B, Corey-Pauling-Koltun view of the core domain of rHGL (yellow) and its cap domain (blue). The putative lid is given by a Cα trace (green).View Large Image Figure ViewerDownload (PPT)Figure 6Stereo view of the interactions of the HPL C11-alkyl phosphonate inhibitor (9Egloff M.-P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (258) Google Scholar) with rHGL . For the C11-phosphonate enantiomer the identified hydrophobic cleft extends beyond the C11 carbon atom by about 7 additional atoms (mimicking a stearoyl chain) and involves on each side: F203, V198, L202 and F212, F207, I206, F205, respectively.View Large Image Figure ViewerDownload (PPT)Figure 5Molecular surface representations of rHGL . A, the complete enzyme; B, same as inA but with the putative lid depleted and the C11-phosphonate enantiomers (red) of HPL represented in the active site (9Egloff M.-P. Marguet F. Buono G. Verger R. Cambillau C. van Tilbeurgh H. Biochemistry. 1995; 34: 2751-2762Crossref PubMed Scopus (258) Google Scholar). Hydrophobic residues are colored blue.View Large Image Figure ViewerDownload (PPT) After numerous trials using weakly diffracting crystal forms obtained from the native gastric lipases purified over the last 10 years from humans, rabbit, and dog (18Moreau H. Abergel C. Carrière F. Ferrato F. Fontecilla-Camps J.C. Cambillau C. Verger R. J. Mol. Biol. 1992; 225: 147-153Crossref PubMed Scopus (37) Google Scholar), useful cubic crystals were finally obtained from rHGL expressed in the insect cell/baculovirus expression system. The structure of rHGL has been solved by a combination of SIRAS, solvent flattening, and 2-fold averaging at 4.5 Å resolution. Phase extension procedures have made it possible to extend the resolution to 3.6 Å. A preliminary model was constructed at this resolution. The final structure was refined to 3.0-Å resolution using restrained noncrystallographic symmetry between the two molecules. The crystallographic R-factor based on all the data between 15.0 and 3.0 Å is 22.6%, and the corresponding R-free is 25.1% with a model containing residues 9 to 53 and 57 to 379, 6 sugar residues located on the 4 potential N-glycosylation sites, and 46 water molecules/monomer. rHGL consists of one globular domain (Fig.1) and belongs to the α/β hydrolase-fold family (1Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Sussman J.L. Verschueren K.H.G. Goldman A. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1835) Google Scholar). The core domain, which is located between residues 9–183 and 309–379 (Figs. 1 and 2) contains a central β sheet composed of 8 strands, 7 of which are parallel and 1 antiparallel (strand 2) with 1(-2)435678 connectivity, and 6 helices, 3 on each side of the β-sheet (Fig. 1 C). Two segments are missing in the electron density maps. The first of these was from residues 4 to 9, because the electron density starts abruptly at residue 9. In the region of the second lacking segment (residues 54 to 56), some faint electron density was observed, suggesting that the loop may be intact but disordered (Fig. 1). The accessibility of this loop is consistent with the previously reported preferential trypsin cleavage site (Arg-55) of rabbit gastric lipase (35De Caro J. Verger R. De Caro A. Biochim. Biophys. Acta. 1998; 1386: 39-49Crossref PubMed Scopus (7) Google Scholar). In comparison with the canonical α/β hydrolase fold, an extra helix (α1) is present at the N terminus. Helix αA is shorter and has moved to the bottom of the structure, helix αB is replaced by two helices (αB1 and αB2), and helix αD is replaced by an extra domain (residues 184 to 308, Figs. 1 and 2) located between strands 6 and 7. Protrusions have been observed in other lipases, generally constituting the device covering the active site and called the lid. A “cap” domain occurs at the same location in wheat serine carboxypeptidase II (residues 181 to 311, WCSII) (36Liao D.I. Breddam K. Sweet R.M. Bullock T. Remington S.J. Biochemistry. 1992; 31: 9796

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Crystal Structure of Human Gastric Lipase and Model of Lysosomal Acid Lipase, Two Lipolytic Enzymes of Medical Interest