Crystal Structure of Human Plasma Platelet-activating Factor Acetylhydrolase
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m804750200
ISSN1083-351X
AutoresUttamkumar Samanta, Brian J. Bahnson,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoHuman plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5Å. It has a classic lipase α/β-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase. Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5Å. It has a classic lipase α/β-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) 2The abbreviations used are: PAF, platelet-activating factor; DEP, diethyl phosphoryl; AH, acetylhydrolase; PLA2, phospholipase A2; HDL, high density lipoprotein; LDL, low density lipoprotein; MOPS, 4-morpholinepropanesulfonic acid; RMSD, root mean square deviation; OPM, orientation of proteins in membranes; MAPAS, membrane associated protein assessment. is a phospholipid messenger synthesized by a variety of cells involved in host defense, such as endothelial cells, platelets, neutrophils, monocytes, and macrophages (1Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1990; 265: 17381-17384Abstract Full Text PDF PubMed Google Scholar). High levels of PAF are responsible for a variety of human diseases such as inflammation, asthma, necrotizing enterocolitis, and sepsis (2Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (593) Google Scholar). The enzyme PAF-AH (EC 3.1.1.47) was first identified from the plasma by its ability to hydrolyze and therefore inactivate PAF (3Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A. Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (481) Google Scholar). The enzyme plasma PAF-AH has been classified as group VIIA phospholipase A2 (PLA2) (4Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (743) Google Scholar), and it hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. In addition to its role in reducing PAF levels, PAF-AH functions by hydrolyzing other pro-inflammatory agents, such as oxidized lipids of LDL particles (5Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar, 6Davis B. Koster G. Douet L.J. Scigelova M. Woffendin G. Ward J.M. Smith A. Humphries J. Burnand K.G. Macphee C.H. Postle A.D. J. Biol. Chem. 2008; 283: 6428-6437Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Many of these oxidized phospholipids have an oxidatively fragmented sn-2 chain that would orient away from the hydrophobic portion of an LDL particle. These oxidized phospholipid species are present at elevated levels at atherogenic lesions, and the PAF-AH hydrolysis of these species has attracted considerable attention recently as a potential therapeutic target (6Davis B. Koster G. Douet L.J. Scigelova M. Woffendin G. Ward J.M. Smith A. Humphries J. Burnand K.G. Macphee C.H. Postle A.D. J. Biol. Chem. 2008; 283: 6428-6437Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 7Macphee C.H. Nelson J.J. Eur. Heart J. 2005; 26: 107-109Crossref PubMed Scopus (11) Google Scholar, 8Macphee C.H. Nelson J. Zalewski A. Curr. Opin. Pharmacol. 2006; 6: 154-161Crossref PubMed Scopus (58) Google Scholar, 9Aiyar N. Disa J. Ao Z.H. Ju H.S. Nerurkar S. Willette R.N. Macphee C.H. Johns D.G. Douglas S.A. Mol. Cell Biochem. 2007; 295: 113-120Crossref PubMed Scopus (109) Google Scholar, 10Wilensky R.L. Shi Y. Zalewski A. Mohler E.R. Hamamdzic D. Li J.U. Pelcovitz D. Webb C. Burgett M.E. Walker M.C. Macphee C.H. Circulation. 2007; 116 (33): 33Google Scholar). Physiologically, plasma PAF-AH is associated to both LDL and HDL particles and therefore functions on the lipid-aqueous interface and can be considered a peripheral membrane protein. Another generally used name for plasma PAF-AH is lipoprotein-associated PLA2 (7Macphee C.H. Nelson J.J. Eur. Heart J. 2005; 26: 107-109Crossref PubMed Scopus (11) Google Scholar). Kinetic studies have shown that although PAF-AH binds to interfaces, such as LDL particles and vesicles, this binding is not necessary for catalysis (11Min J.H. Jain M.K. Wilder C. Paul L. Apitz-Castro R. Aspleaf D.C. Gelb M.H. Biochemistry. 1999; 38: 12935-12942Crossref PubMed Scopus (65) Google Scholar, 12Min J.H. Wilder C. Aoki J. Arai H. Inoue K. Paul L. Gelb M.H. Biochemistry. 2001; 40: 4539-4549Crossref PubMed Scopus (64) Google Scholar). This is in contrast to the 14-kDa secreted PLA2 enzymes that are allosterically activated upon interface binding (13Gelb M.H. Min J.H. Jain M.K. Biochim. Biophys. Acta. 2000; 1488: 20-27Crossref PubMed Scopus (54) Google Scholar). The plasma PAF-AH and the homologous (42% identity) intracellular type II PAF-AH (also known as group VIIB PLA2) enzymes are calcium-independent and contain a GXSXG motif that is characteristic of neutral lipases and serine esterases (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Structurally distinct from the 44-kDa plasma PAF-AH and type II PAF-AH enzymes is a 26-kDa intracellular brain catalytic domain that has been designated type I PAF-AH or more commonly PAF-AH-Ib (15Arai H. Prostaglandins Other Lipid Mediat. 2002; 68–69: 83-94Crossref PubMed Scopus (70) Google Scholar). Although the structure of PAF-AH-Ib has been reported (16Ho Y.S. Swenson L. Derewenda U. Serre L. Wei Y. Dauter Z. Hattori M. Adachi T. Aoki J. Arai H. Inoue K. Derewenda Z.S. Nature. 1997; 385: 89-93Crossref PubMed Scopus (159) Google Scholar, 17Tarricone C. Perrina F. Monzani S. Massimiliano L. Kim M.H. Derewenda Z.S. Knapp S. Tsai L.H. Musacchio A. Neuron. 2004; 44: 809-821Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), there is no sequence homology to the other PAF-AH enzymes. Additionally, the PAF-AH-Ib enzyme has been suggested to have a physiological function other than as a true PAF-AH enzyme (18Derewenda Z.S. Colloids Surfaces B: Biointerfaces. 2002; 26: 31-35Crossref Scopus (3) Google Scholar, 19Epstein, T. M. (2005) Structural and Kinetic Studies of Two Enzymes Catalyzing Phospholipase A2 Activity. Ph.D. thesis, University of Delaware, Newark, DEGoogle Scholar). Although a structure has been determined for a distantly related lipase from Streptomyces exfoliatus (20Wei Y. Swenson L. Castro C. Derewenda U. Minor W. Arai H. Aoki J. Inoue K. Servin-Gonzalez L. Derewenda Z.S. Structure. 1998; 6: 511-519Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), the extent of sequence overlap between this enzyme and the mammalian plasma PAF-AH enzyme is limited to 19% identity over a subset of 228 aligned residues. Hence, a crystal structure of a mammalian PAF-AH was required to understand the relationship between structure and function. Following identification of the enzyme in the plasma, it had been subjected to extensive biochemical characterization, including the determination of the limits of N- and C-terminal truncations that preserve native functions (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Here, we report a 1.5-Å ligand-free crystal structure and a 2.1-Å diethyl phosphoryl (DEP) complex crystal structure of a 43.4-kDa construct (residues 47–429) of PAF-AH. This construct represents a form of the enzyme with native-like enzyme activity (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) toward the substrate PAF, as well as native-like LDL binding properties (12Min J.H. Wilder C. Aoki J. Arai H. Inoue K. Paul L. Gelb M.H. Biochemistry. 2001; 40: 4539-4549Crossref PubMed Scopus (64) Google Scholar). Protein Preparation and Crystallization—We obtained 160 mg of pure human plasma PAF-AH (PAFase, residues 47–429, NCBI accession Q13093) overexpressed from Escherichia coli from ICOS Corporation. Details of the protein preparation and initial crystallization of the ligand-free form of human PAF-AH have been reported (21Samanta, U., Wilder, C., and Bahnson, B. J. (2008) Protein Peptide Lett., in pressGoogle Scholar) and will be summarized briefly. Prior to crystallization, PAF-AH samples were suspended in appropriate buffer/detergent solutions (see below). PAF-AH was assayed for kinetic activity using the ester substrate p-nitrophenyl acetate, and the reaction was followed by UV absorbance at 402 nm (ϵ402 nm = 17,700 m-1cm-1). Ligand-free PAF-AH Crystals—Our best crystals of ligand-free PAF-AH were obtained at 20 °C starting from a protein solution at 4 mg/ml that contained 10 mm Tris-HCl, 6 mm sodium citrate, 3% (w/v) sucrose, 1.0 mm dithiothreitol, 27 mmn-octyl-β-d-glucopyranoside, 0.04% (w/v) Pluronic F68, 0.002% (w/v) Tween 80, pH 6.7. The crystallization reservoir solution contained 98.5 mm MOPS buffer, pH 6.6, 44.3% (w/v) (NH4)2SO4, 0.394 m Li2SO4, 0.985 m sodium acetate, and 1.48% (v/v) 1,4-butanediol. Aliquots of 1.5 μl of protein and crystallization solutions were mixed to form each hanging drop, and protein crystals formed in 3–4 weeks. To obtain phase information, the ligand-free crystals of PAF-AH were directly soaked with MeHgCl by adding a small grain of solid powder to the hanging drop and incubating it in the drop for 24 h. Derivative crystals were then flash cooled in liquid nitrogen, stored, and transported to the synchrotron for data collection. Paraoxon Inhibited PAF-AH—PAF-AH protein received from ICOS Corp. was exchanged with the detergent Triton DF16 (0.01% w/v) using a Q-Sepharose column as described previously (21Samanta, U., Wilder, C., and Bahnson, B. J. (2008) Protein Peptide Lett., in pressGoogle Scholar). The protein was then incubated overnight with the organophosphate compound paraoxon (ChemService) to covalently react with the enzyme active site Ser273. Protein was dialyzed to remove salt and excess paraoxon, and finally it was assayed to confirm a complete loss of enzyme activity. The protein sample was concentrated by centrifugal concentration using a Centricon 30 membrane (Millipore) at slow speed (2000 rpm) to avoid aggregation and precipitation. The protein component contained 3 mg/ml protein, 1.0 mm dithiothreitol, 27 mmn-octyl-β-d-glucopyranoside. The crystallization solution contained 41% (w/v) (NH4)2SO4, 400 mm Li2SO4, 800 mm sodium formate, pH 7.0, 1.24% (w/v) 1,4-butanediol. Aliquots of 1.5 μl of the protein and crystallization solutions were mixed and set as hanging drops. The crystals grew (150 × 120 × 90 μm) within 2 weeks. X-ray Data Collection—Native and mercury-derivative data sets were collected at synchrotron beamlines using crystals frozen in liquid nitrogen; additional cryo-protectant was not required. The single- and multiple-wavelength anomalous dispersion data sets were collected at Beamline 19ID of the Advanced Photon Source (Argonne National Laboratory, Chicago, IL). The data sets for native ligand-free and paraoxon inhibited crystals were collected at Beamline X29 of the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY). The diffraction data were indexed and processed using the program HKL2000 (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar). Crystal Structure Solution—A single-wavelength anomalous dispersion data set to a resolution of 2.71 Å of a MeHgCl derivative of PAF-AH was used to phase the structure. Eight mercury sites in the crystallographic asymmetric unit were identified and used to generate initial phases using the programs SOLVE and RESOLVE (23Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (435) Google Scholar). Two subunits of PAF-AH are in the asymmetric unit. A noncrystallographic symmetry axis was identified from six of the mercury sites, with three similar sites per protein subunit. The noncrystallographic symmetry axis was used to improve initial electron density maps using the CCP4 suite program Dm (24CCP4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). These medium resolution electron density maps, which contained regions of well defined α-helices and β-sheets, allowed the initial model building of roughly half the 766 amino acids/asymmetric unit with the program COOT (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (24326) Google Scholar). The partial model was then refined in the nonisomorphous native data set using data to a resolution of 2.6 Å. The partial model was next used as initial phases together with the native data set to a resolution of 1.5 Å for the automated program ARP/wARP (26Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2571) Google Scholar). Within 60 cycles, the program had traced and auto-built side chains for 97% of the model. Refinement was initially carried out with the program REFMAC5 (24CCP4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar) and was finished using the program SHELXL (27Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1903) Google Scholar). Structure of PAF-AH-DEP Complex—A single subunit of the ligand-free PAF-AH structure was used as a search model. Molecular replacement was performed using the program MOLREP (24CCP4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar) to solve the structure of the PAF-AH-DEP complex, which had been crystallized using paraoxon inhibited PAF-AH. The structure of the PAF-AH-DEP complex was refined using the program REFMAC5 (24CCP4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). A CIF parameter file for the DEP group, which is covalently attached to Ser273, was prepared using the monomer library sketcher module of the program CCP4 (24CCP4.Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). Overall Structure of PAF-AH—We have solved the crystal structures of human plasma PAF-AH in a ligand-free form and as a complex with the organophosphate compound paraoxon reacted with the active site Ser273 (Fig. 1). The protein displays a classic α/β-hydrolase fold, typical of other GXSXG lipases. The structure of plasma PAF-AH was submitted to the DALI website to obtain structural homologues (28Holm L. Sander C. Science. 1996; 273: 595-602Crossref PubMed Scopus (1296) Google Scholar); the S. exfoliatus lipase structure (20Wei Y. Swenson L. Castro C. Derewenda U. Minor W. Arai H. Aoki J. Inoue K. Servin-Gonzalez L. Derewenda Z.S. Structure. 1998; 6: 511-519Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) was the top hit with a Z score of 23.8, Cα RMSD of 2.4 Å, and 19% identity over a subset of 228 amino acids aligned. The topology diagram of the PAF-AH structure is shown in supplemental Fig. S1. In both of the crystal structures reported here, there are two protein subunits in the asymmetric unit. The construct of PAF-AH crystallized contained 383 residues (47–429). A summary of the data collection, phasing and refinement of the crystal structures is presented in Table 1.TABLE 1Data collection, phasing, and refinement statistics of PAF-AH The values in parentheses are for the highest resolution shell. The RMSD bond angles are reported as angle distances using the program SHELXL and as degrees in the program REFMAC5.MeHgClNativeDEP complexCrystal parametersSpace groupC2C2C2Unit cell dimensions a, b, c (Å), β(°)114.7, 79.0, 96.5, 115.3116.2, 83.1, 96.7, 115.1117.2, 78.7, 97.3, 101.6BeamlineAPS-19IDNSLS-X29NSLS-X29Data collectionPeakWave length (Å)1.006aThese are the peak values1.1001.072Resolution (Å)50-2.7 (2.8-2.7)aThese are the peak values50-1.5 (1.55-1.50)50-2.1 (2.18-2.10)Completeness (%)98.6 (97.4)aThese are the peak values96.4 (75.7)94.9 (73.8)Redundancy2.1 (2.1)aThese are the peak values3.6 (1.8)3.4 (2.1)I/σI18.3 (2.1)aThese are the peak values19.2 (1.9)19.6 (3.4)Rmerge linear0.047 (0.387)aThese are the peak values0.057 (0.283)0.053 (0.193)Phasing statisticsNumber of mercury sites8aThese are the peak valuesPhasing power anomalous0.603aThese are the peak valuesR factor (FC vs. FP)bThe R factor compares structure factors calculated from density modification (FC) to those measured (FP)0.298aThese are the peak valuesFigure of merit (acentric/centric/all)0.70/0.77/0.71aThese are the peak valuesRefinement statisticsRefinement programSHELXLREFMAC5Resolution (Å)10.0-1.550.0-2.1Rwork/Rfree0.131/0.1790.208/0.264Number of atoms (non-hydrogen)66926366B-factor main chain24.028.3B-factor side chain31.331.1RMSD bond lengths (Å)0.010.02RMSD bond angles0.03 Å1.87°Ramachandran plot (most favored)91.6%89.5%Ramachandran plot (most favored and additionally allowed region)99.5%99.7%a These are the peak valuesb The R factor compares structure factors calculated from density modification (FC) to those measured (FP) Open table in a new tab Quality of the Structures—As expected, the higher resolution 1.5 Å ligand-free structure has better statistics than the 2.1 Å DEP complex structure. The Ramachandran plots of the ligand-free complex and the DEP complex had 91.6 and 89.5% of residues in the most favored region, respectively. Only residue Ser273, which is the active site nucleophile, was observed to be outside of the generously allowed region (phi, 65; psi, -116). The electron density of this region, as shown for the DEP complex structure in Fig. 1D, is very distinct, supporting its proper modeling. The same holds true for the electron density difference map of the 1.5 Å ligand-free structure. The strained conformation of this serine is consistent with other α/β hydrolase enzymes that have nucleophilic serines in a strained ϵ-conformation (29Ollis 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 (1875) Google Scholar). The overall B-factor and deviations from ideal geometry are likewise slightly smaller in the ligand-free structure. One other noteworthy outlier in each subunit of both structures reported is the presence of a cis-peptide bond between Phe72 and Asp73. The electron density of this region is very distinct, thereby supporting the presence of this unusual cis-peptide bond. Disordered Residues and Side Chains—The N- and C-terminal limits of ordered residues observed in our crystal structures are consistent with functional observations of the protein. Plasma PAF-AH functions as a 43-, 44-, or 45-kDa protein with a heterogeneous N terminus starting at residue Ser35, Ile42 or Lys55 (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In our structures, seven residues (positions 47–53) are missing from the N terminus in each subunit of both the ligand-free and DEP-bound structures. In each case, our structures begin with residue 54, which is close to the endogenous N-terminal start site at Lys55. In previous studies, the limits of N- and C-terminal truncations had been characterized that still ensure the presence of the full native functions of the enzyme (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In our crystal structures, the C terminus has several disordered residues in the ligand-free structure (A, 426–429; B, 428–429) and DEP complex (A, 427–429; B, 424–429). The ordered C-terminal end of our structures is close to the functional limit tolerated by C-terminal truncation. A subtle difference exists between the two subunits of the ligand-free crystal structure in the crystallographic dimer. In subunit B the electron density of residues 88–92 was weak; therefore their side chain atoms have been truncated to correspond to an alanine side chain. Similarly His114 and Trp115 are missing in subunit A and Trp115 and Leu116 are missing in subunit B; therefore each of these side chains have been truncated as well. In addition to these residues mentioned above, several other surface-accessible and polar side chains from the ligand-free crystal structure were disordered and therefore not modeled. In contrast, the DEP-bound crystal structure had well ordered side chain positions for all residues modeled, including residues His114–Leu116. Interface Binding Surface of PAF-AH—The disorder of the residues His114–Leu116, described above, can be explained because of the flexibility of these residues on the surface of the protein. Additionally, these residues are on an α-helix (114–126) that is predicted to be a component of the interfacial binding surface of the protein that accesses the lipoprotein particle. Specific residues shown to be important for binding to LDL by site-directed mutagenesis were Tyr205, Trp115, Leu116, and to a lesser extent Met117 (30Stafforini D.M. Tjoelker L.W. McCormick S.P. Vaitkus D. McIntyre T.M. Gray P.W. Young S.G. Prescott S.M. J. Biol. Chem. 1999; 274: 7018-7024Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). A second short helix (residues 362–369) has hydrophobic residues positioned to insert into the hydrophobic portion of the aqueous-lipid interface. Recently, residues near this helix were predicted to be important for HDL binding (31Gardner A.A. Reichert E.C. Topham M.K. Stafforini D.M. J. Biol. Chem. 2008; 283: 17099-17106Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The predicted interfacial-binding residues of PAF-AH are displayed in Fig. 1B relative to the active site. Active Site of PAF-AH—As originally predicted (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 32Derewenda Z.S. Derewenda U. Cell. Mol. Life Sci. 1998; 54: 446-455Crossref PubMed Scopus (25) Google Scholar) the active site of PAF-AH contains a catalytic triad of Ser273, His351, and Asp296. Ser273 is located at the N terminus of an α-helix and on the conserved GXSXG motif found in other lipases (14Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Prescott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The amide nitrogens of Phe274 and Leu153 are well poised to stabilize the negative charge of a tetrahedral intermediate of the reaction mechanism, thereby acting as the oxyanion hole of the enzyme. The other two catalytic triad residues are appropriately positioned to activate the nucleophilic Ser273 for catalysis; Asp296 is located on the C-terminal end of a β-sheet, and His351 is located at the N terminus of an α-helix. Like other lipases, the catalytic triad lies within a hydrophobic pocket that is oriented toward its substrate (Fig. 1, B and C). Notably, the orientation of the catalytic triad and hydrophobic residues Leu153 and Phe274 were previously predicted based on modeling from the distantly related S. exfoliatus lipase structure (32Derewenda Z.S. Derewenda U. Cell. Mol. Life Sci. 1998; 54: 446-455Crossref PubMed Scopus (25) Google Scholar). From previous functional and kinetic characterization of PAF-AH, it was suggested that the active site would allow substrates to enter from lipoproteins and the aqueous phase (11Min J.H. Jain M.K. Wilder C. Paul L. Apitz-Castro R. Aspleaf D.C. Gelb M.H. Biochemistry. 1999; 38: 12935-12942Crossref PubMed Scopus (65) Google Scholar, 12Min J.H. Wilder C. Aoki J. Arai H. Inoue K. Paul L. Gelb M.H. Biochemistry. 2001; 40: 4539-4549Crossref PubMed Scopus (64) Google Scholar). The placement of the active site observed in the structure is consistent with these previous findings. DEP Complex of PAF-AH—To gain insights into the active site properties of PAF-AH, we solved the crystal structure of a covalent complex with the organophosphate compound paraoxon. The enzyme was quickly inactivated by paraoxon, as determined by enzymatic assay. Crystal set-ups led to crystals in the C2 space group, just as with the ligand-free structure, but with slightly different cell dimensions (Table 1). An overall alignment of the ligand-free and DEP complex crystal structures shows only subtle differences of side chain positions. A comparison of the ligand-free and DEP complex structures shows an overall Cα RMSD of 0.3 Å. The active site residues are virtually unchanged, aside from the covalent attachment of DEP to Ser273 as depicted in Fig. 2; the complex with DEP-bound (panel A) serves as a mimic of the tetrahedral intermediate of the esterolysis reaction (panel B). The catalytic triad residues and neighboring active site residues are positioned around the PAF-AH-DEP complex in a manner consistent with a tetrahedral intermediate complex (Fig. 1D). Contact distances between active site residues and the bound DEP group are shown in Table 2. Notably, the amide nitrogens of Phe274 and Leu153 make H-bonds with the O-3 oxygen of the DEP moiety, which would correspond with the enolate oxygen of a tetrahedral intermediate. The positions of the two ethoxy groups of the bound DEP group are well ordered, as shown in the electron density maps shown in Fig. 1D. A view of these two groups are shown relative to the active site pocket, which is rendered with a space filling electrostatic surface in Fig. 1C. This complex will be further discussed below, as a means to begin to understand how substrates bind to PAF-AH.TABLE 2DEP and surrounding active site residue contacts within 4 Å Hydrogen bond contacts are shown in italics.DEP atomResidueAtom typeDistanceÅPHis351NE23.74O-1Trp298CE33.41O-2His351NE23.08O-3Gly152C3.58Leu153N2.65Phe274N2.90C-1Phe322CZ3.62C-2Trp298CE23.69C-3Leu153O3.18His351NE23.93C-4Leu153O3.72His272CE13.80His351NE23.89 Open table in a new tab Two Subunits in Asymmetric Unit—Although PAF-AH does not show any oligomeric association in solution, the crystals contain two subunits in the asymmetric unit. A comparison of the A and B subunits shows a Cα RMSD of 0.3 Å for the ligand-free s
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