SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2053104328> ?p ?o ?g. }

Showing items 1 to 100 of ±117 with 100 items per page.

W2053104328 endingPage "3085" @default.
W2053104328 startingPage "3076" @default.
W2053104328 abstract "Impairment of the formation or action of hydrogen sulfide (H2S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine β-synthase and cystathionine γ-lyase (CSE) are two pyridoxal-5′-phosphate (PLP)-dependent enzymes largely responsible for the production of H2S in mammals. Inhibition of CSE by dl-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP·PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H2S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE·PLP·PAG complex highlights the particular importance of Tyr114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism. Impairment of the formation or action of hydrogen sulfide (H2S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine β-synthase and cystathionine γ-lyase (CSE) are two pyridoxal-5′-phosphate (PLP)-dependent enzymes largely responsible for the production of H2S in mammals. Inhibition of CSE by dl-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP·PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H2S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE·PLP·PAG complex highlights the particular importance of Tyr114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism. Gaseous messengers or gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have been shown to be important in a range of biological systems. In particular, H2S has recently gained interest as a mediator of cardiovascular and nervous system functions and in inflammation (1Szabo C. Nat. Rev... 2007; 6: 917-935Google Scholar, 2Li L. Bhatia M. Moore P.K. Curr. Opin. Pharmacol... 2006; 6: 125-129Google Scholar, 3Li L. Moore P.K. Trends Pharmacol. Sci... 2008; 29: 84-90Google Scholar). Numerous organisms, including bacteria, archaea, and nonmammalian vertebrates as well as mammals, have been shown to produce and utilize H2S gas as signaling molecule at physiological concentrations (20–160 μm) (4Zhao W. Zhang J. Lu Y. Wang R. EMBO J.. 2001; 20: 6008-6016Google Scholar). In mammals, two pyridoxal-5′-phosphate (PLP) 5The abbreviations used are: PLP, pyridoxal-5′-phosphate; CSE, cystathionine γ-lyase; hCSE, human CSE; PAG, dl-propargylglycine; GST, glutathione S-transferase; TLS, translation/libration/screw.5The abbreviations used are: PLP, pyridoxal-5′-phosphate; CSE, cystathionine γ-lyase; hCSE, human CSE; PAG, dl-propargylglycine; GST, glutathione S-transferase; TLS, translation/libration/screw.-dependent enzymes, cystathionine β-synthase and cystathionine γ-lyase (CSE), are largely responsible for the in vivo production of H2S. Cystathionine β-synthase is expressed predominantly in the central nervous system, and the regulation of cystathionine β-synthase has been well studied (5Miles E.W. Kraus J.P. J. Biol. Chem... 2004; 279: 29871-29874Google Scholar), whereas CSE is mainly responsible for the production of H2S outside of the nervous system (3Li L. Moore P.K. Trends Pharmacol. Sci... 2008; 29: 84-90Google Scholar), and its regulatory mechanisms are less well understood. Natural, nonactive CSE mutations, such as T67I and Q240E (6Zhu W. Lin A. Banerjee R. Biochemistry.. 2008; 47: 6226-6232Google Scholar), are associated with cystathioninuria, a disease condition characterized by accumulation of cystathionine in blood, tissue, and urine, sometimes also associated with mental retardation (7Tang C. Li X. Du J. Curr. Vasc. Pharmacol... 2006; 4: 17-22Google Scholar). Two of these disease-linked mutations have recently been studied and shown to weaken affinity for PLP (6Zhu W. Lin A. Banerjee R. Biochemistry.. 2008; 47: 6226-6232Google Scholar). Moreover, induction of endotoxemia, acute pancreatitis, hemorrhagic shock, pulmonary hypoxic hypertension, and diabetes mellitus in animals is associated with increased H2S production due to up-regulated CSE production (1Szabo C. Nat. Rev... 2007; 6: 917-935Google Scholar, 2Li L. Bhatia M. Moore P.K. Curr. Opin. Pharmacol... 2006; 6: 125-129Google Scholar). Further, work using several of these animal disease models have revealed that either pre- or post-treatment of animals with inhibitors of CSE, such as dl-propargylglycine (PAG) or β-cyanoalanine, not only inhibit tissue H2S production but also reduce the severity of the disease state. Albeit carried out in animals, these studies serve to highlight the potential importance of CSE-mediated production of H2S in regulating a number of physiological processes in humans. Steegborn et al. (27Steegborn C. Clausen T. Sondermann P. Jacob U. Worbs M. Marinkovic S. Huber R. Wahl M.C. J. Biol. Chem... 1999; 274: 12675-12684Google Scholar) studied the kinetics of hCSE and reported the activity of this enzyme toward l-cystathionine, l-cystine, and l-cysteine, and several inhibition studies were performed with CSE inhibitors, including PAG. Structure of the Saccharomyces cerevisiae homolog of hCSE revealed insights into the enzymatic specificity among the different family members (9Messerschmidt A. Worbs M. Steegborn C. Wahl M.C. Huber R. Laber B. Clausen T. Biol. Chem... 2003; 384: 373-386Google Scholar). Recently, Yang et al. (34Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. Snyden S.H. Wang R. Science.. 2008; 322: 587-590Google Scholar) showed that H2S is physiologically generated by CSE and that genetic deletion of CSE in mice markedly reduces H2S levels in the serum, heart, aorta, and other tissues. Mutant mice lacking CSE display significant hypertension and reduced endothelium-dependent vasorelaxation and provide direct evidence that H2S is a physiologic vasodilator and regulator of blood pressure. We report here crystal structures of human cystathionine γ-lyase in the apo form (apo-hCSE), complexed with PLP (hCSE·PLP) and with the inhibitor PAG (hCSE·PLP·PAG) at 2.4, 2.6, and 2.0 Å resolutions, respectively. This is the first report of any crystal structure of a PLP-dependent enzyme in an open conformation that does not have PLP (or a PLP derivative) bound in the active site. In addition, when compared with other PAG complexes, the PAG in hCSE reveals a unique inhibition mechanism, in which PAG does not bind to PLP but adopts a new position in the active site and highlights the singular importance of Tyr114 in hCSE. Further, the crystal structures together with the biochemical and biophysical characterizations provide deeper understanding of the mechanism of hCSE catalysis. We believe that these findings will assist the development of novel inhibitors of hCSE that may be of clinical use in the treatment of a range of disease states resulting from overproduction of H2S. Cloning, Expression, and Purification—The hCSE gene was cloned into a pET-28-based expression vector incorporating a tobacco etch virus-cleavable N-terminal His tag fusion (pNIC-Bsa4). The plasmid was transformed into Escherichia coli BL-21(DE3) and grown in a potassium phosphate-buffered Terrific Broth medium. Expression of hCSE was induced by the addition of 0.5 mm isopropyl β-d-1-thiogalactopyranoside, and the culture was grown overnight at 18 °C. Cells were harvested and sonicated in lysis buffer (50 mm sodium phosphate, pH 7.5, 10% glycerol, 0.5 mm tris(2-carboxyethyl)phosphine, 300 mm NaCl, 10 mm imidazole, and Complete EDTA-free protease inhibitor (Roche Applied Science). hCSE was purified on a Hi-Trap chelating nickel column (elution buffer: 50 mm sodium phosphate, 10% glycerol, 0.5 mm tris(2-carboxyethyl)phosphine, 300 mm NaCl, 500 mm imidazole, pH 7.5), followed by a Superdex 200 gel filtration column on the Äkta Express (GE Healthcare) using the gel filtration buffer (20 mm HEPES, 300 mm NaCl, 10% glycerol, 0.5 mm tris(2-carboxyethyl)phosphine, pH 7.5). The N-terminal hexahistidine tag was removed by incubating hCSE protein with His-tagged tobacco etch virus protease at a ratio of 30:1 and passing the cleavage reaction through a HisTrap FF crude column in gel filtration buffer supplemented with 10 mm imidazole. Eluted hCSE was concentrated to 12.2 mg/ml. At the same time, hCSE cDNA was cloned into pGEX4T-3 to create an N-terminal GST fusion. The expression vector was transformed into E. coli BL21 and induced at 0.1 mm isopropyl β-d-1-thiogalactopyranoside at 18 °C overnight. Cells were harvested, sonicated, and lysed in 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 5 mm dithiothreitol, and the protease inhibitor mixture (Sigma). hCSE protein was cleaved in the GST beads with thrombin and eluted from the beads with 20 mm sodium phosphate, pH 7.8, 5 mm β-mercaptoethanol, 50 units of thrombin (Sigma). The eluted protein was further passed through a Superdex 200 gel filtration column using an ÄKTA FPLC UPC-900 system (GE Healthcare) and was concentrated to 5 mg/ml in 10 mm sodium phosphate, pH 8.2, and 1 mm dithiothreitol. It has to be noted that this work was initially started independently by two different research groups using two different expression systems. Crystallization and Data Collection—Initial crystallization conditions were identified from the JCSG+ and Nextal crystal screens (Qiagen) using sitting drop and hanging drop vapor diffusion methods at room temperature. Crystals were grown by mixing equal amounts of protein solution (10 mg/ml hCSE·PLP complex; 5 mg/ml apo-hCSE in the presence of 10 mm l-cysteine) and reservoir solution containing 15% (v/v) polyethylene glycol 3350, 200 mm ammonium citrate, pH 5.6, for hCSE·PLP complex and 20% (v/v) polyethylene glycol 1000, 0.2 m lithium sulfate, 0.1 m phosphate-citrate, pH 4.2, for the apo-hCSE. All tags were removed prior to crystallization. Regardless of their fusion tags, the proteins used for crystallization experiments were equivalent and showed the characteristic absorption for the PLP-lysine internal aldimine bond. However, the bound PLP of the GST-tagged purified hCSE was most likely lost during crystallization. For the apo-hCSE, rod-shaped crystals with the smallest dimension measuring ∼30 μm were obtained after 3 days. X-ray diffraction data were collected at beamline X12C (National Synchrotron Light Source, Brookhaven National Laboratory) using a Q315 CCD detector (Area Detector Systems Corp., Poway, CA). Plate like crystals of hCSE·PLP appeared after 1 day and continued to grow for 1 week. The hCSE·PLP·PAG complex was obtained by soaking crystals of hCSE·PLP in mother liquid supplemented with 10 mm PAG, incubating for 55 min. Diffraction data for hCSE·PLP was collected at the European Synchrotron Radiation Facility beamline ID29 (Grenoble, France) on a Q315 CCD detector (Area Detector Systems Corp., Poway, CA). For the hCSE·PLP·PAG complex, diffraction data were collected at Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY) beamline BL 14.1 (Berlin, Germany) on a MarCCD detector. 20–25% (v/v) glycerol was supplemented with crystallization condition as the cryoprotectant for all crystals used in the experiments. The crystallographic statistics are given in Table 1.TABLE 1Crystallographic data and refinement statisticsData setapo-hCSEhCSE·PLPhCSE·PLP·PAGData collectionCell axial lengths (Å)121.31, 121.31, 125.59105.78, 107.57, 153.31105.35, 107.22, 153.31Space groupP42P212121P212121Resolution range (Å)40.0–2.4 (2.5–2.4)30.0–2.6 (2.7–2.6)20.0–2.0 (2.1–2.0)Wavelength (Å)0.97921.03320.9184Observed reflections625250399157732975Unique reflections7098554383109200Completeness (%)99.8 (98.8)99.8 (99.3)92.9 (98.9)Overall (I/σI)14.2 (2.1)7.2 (3.4)13.8 (3.2)RsymaRsym = Σ|Ii – 〈I〉|/Σ|Ii|, where Ii is the intensity of the ith measurement, and 〈I〉 is the mean intensity for that reflection (%)0.127 (0.676)0.140 (0.517)0.123 (0.445)Solvent content (%)52.247.249.6Refinement and qualityResolution range (Å) I > σ(I)20.0–2.419.9–2.619.9–2.0RworkbRwork = Σ|Fo – Fc|/|Fo|, where Fc and Fo are the calculated and observed structure factor amplitudes, respectively (no. of reflections)0.221 (66,936)0.180 (51,628)0.158 (98,278)RfreecRfree calculated as for Rwork but for 5.0% of the total reflections chosen at random and omitted from refinement for all data sets (no. of reflections)0.236 (3582)0.245 (2718)0.204 (5460)Root mean square deviation bond lengths (Å)0.0070.0140.013Root mean square deviation bond angles (degrees)1.381.641.37Average B-factors (Å2) (no. of atoms)Protein atoms50.7 (10,633)24.8 (11,846)17.4 (11,875)Ligand atomsPLPNAdNot applicable31.2 (45)16.5 (45)PAG26.5 (24)Water molecules42.9 (468)22.2 (157)27.9 (1159)Ramachandran plotMost favored regions (%)86.289.690.1Additional allowed regions (%)129.59.0Generously allowed regions (%)0.60.60.4Disallowed regions (%)1.30.30.4a Rsym = Σ|Ii – 〈I〉|/Σ|Ii|, where Ii is the intensity of the ith measurement, and 〈I〉 is the mean intensity for that reflectionb Rwork = Σ|Fo – Fc|/|Fo|, where Fc and Fo are the calculated and observed structure factor amplitudes, respectivelyc Rfree calculated as for Rwork but for 5.0% of the total reflections chosen at random and omitted from refinement for all data setsd Not applicable Open table in a new tab Structure Solution and Refinement—The structure of hCSE·PLP complex was solved using the molecular replacement method with the program MolRep (8Vagin A. Teplyakov A. Acta Crystallogr... 1997; 30: 1022-1025Google Scholar) with the coordinates of S. cerevisiae CSE (9Messerschmidt A. Worbs M. Steegborn C. Wahl M.C. Huber R. Laber B. Clausen T. Biol. Chem... 2003; 384: 373-386Google Scholar) (Protein Data Bank code 1N8P) as the search model. All four molecules of the asymmetric unit were identified. The resulting model with the electron density map was examined in the program COOT (10Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr... 2004; 60: 2126-2132Google Scholar), and the necessary manual model building was carried out. Several cycles of map fitting and refinement using the program Refmac5 (11Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr... 1997; 53: 240-255Google Scholar) led to convergence. Translation/libration/screw (TLS) refinement using three TLS groups per monomer were used in the refinement process (12Painter J. Merritt E.A. Acta Crystallogr... 2006; 39: 109-111Google Scholar). The TLS groups were selected using the tlsmd server (available on the World Wide Web). Data in the interval 19.9–2.6 Å resolution were used, and at the end of the refinement the R value was 0.180 (Rfree = 0.245) for all reflections. Similarly, the hCSE·PLP coordinates were used to determine the structure of apo-hCSE by molecular replacement using MolRep (8Vagin A. Teplyakov A. Acta Crystallogr... 1997; 30: 1022-1025Google Scholar). Four molecules of the asymmetric unit were identified. The model building and refinement were carried out using the programs COOT (10Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr... 2004; 60: 2126-2132Google Scholar) and CNS (13Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr... 1998; 54: 905-921Google Scholar), which led to the convergence of R factor to 0.221 (Rfree = 0.236) for reflections with I > σI up to 2.4 Å resolution. For the hCSE·PLP·PAG complex, the coordinates for hCSE·PLP were used to obtain initial phases using MolRep (8Vagin A. Teplyakov A. Acta Crystallogr... 1997; 30: 1022-1025Google Scholar). Four molecules were present in the asymmetric unit. Data in the interval 19.9–2.0 Å resolution were used, and at the end of the refinement, the R value was 0.158 (Rfree = 0.204) for all reflections. For both hCSE·PLP and hCSE·PLP·PAG complexes and apo-hCSE, noncrystallographic symmetry restraints were applied throughout the refinement. Size Exclusion Analysis of hCSE—Purified hCSE was maintained in 10 mm sodium phosphate (pH 7.8) at a concentration of 6 mg/ml. hCSE was then diluted with either buffer A (10 mm sodium phosphate, pH 7.8) or buffer B (50 mm sodium citrate, pH 4.0, 10 mm cysteine) to 0.5 mg/ml and incubated at 4 °C overnight. These two samples were then loaded on 16/60 Superdex 200 column and run with buffer A or B, respectively. Analytical Ultracentrifugation Experiment—The oligomeric state of hCSE was investigated by using a Beckman-Coulter XL-I analytical ultracentrifuge fitted with an eight-hole AN-50 Ti rotor and double-sector centerpieces. Samples used for these experiments were 0.4 ml in volume and a concentration of 0.5 mg/ml in buffer A or buffer B, and the same buffer of 0.44 ml in volume was loaded in the reference sector. The sedimentation velocity profiles were collected by monitoring the absorbance at 280 nm as the samples were centrifuged at 40,000 rpm at 20 °C. Multiple scans at different time points were fitted to a continuous size distribution by using the SEDFIT program (14Schuck P. Biophys. J... 2000; 78: 1606-1619Google Scholar). Ion Mobility Mass Spectrometry Analysis—Ion mobility mass spectrometry analysis was carried out on a Synapt HDMS mass spectrometer with 8K quadrupole (Waters, Manchester, UK). The spectra were displayed in Masslynx, and IMS ToF data were displayed in DriftScope. Samples of PLP-bound hCSE and apo-hCSE (∼2 μm) in buffer A and buffer B were buffer-exchanged into 50 mm ammonium acetate, pH 6.5, and injected using a nanospray source. Assay of H2S Production—Kinetics studies were performed using a spectrophotometric assay as described by Stipanuk and Beck (15Stipanuk M.H. Beck P.W. Biochem. J... 1982; 206: 267-277Google Scholar) with some minor modifications. Each test consisted of 5 μg of the purified hCSE enzyme, 2 mm PLP, and l-cysteine (0.75–3.5 mm) in 1.5-ml cryovial tubes. After incubation at 37 °C, 1% (w/v) zinc acetate was added to trap the evolved H2S gas, and the enzymatic reaction was stopped by the addition of 10% (w/v) trichloroacetic acid. 20 mm N,N-dimethyl-p-phenylenediamine dihydrochloride dye and 30 mm FeCl3 were then added, and the absorbance at 670 nm was measured using a microplate reader (Tecan Systems Inc.). Controls were prepared by adding 10% (w/v) trichloroacetic acid before the addition of cysteine to stop all enzymatic reactions. The amount of H2S produced was determined every 3 min over 30 min and calculated against a calibration curve of sodium hydrosulfide. The initial reaction velocity, V0 (units/mg hCSE, where 1 unit = 1 μmol of H2S produced/min) was then plotted against the cysteine substrate concentration, [S], and fitted against the Hill equation, V0=Vmax[S]hK0.5+[S]h(Eq. 1) The Hill coefficient, h, was determined from the gradient of the logarithmic plot of the Hill equation, log (V0Vmax−V0)=hlog [S]+constant(Eq. 2) All curve fitting and regression analysis were performed using the graphics software SigmaPlot (Systat). IC50 Analysis on PAG—Inhibition of hCSE-dependent H2S production was determined by performing the assay with varying concentrations of PAG. Each measurement was assayed in triplicates. Purified hCSE was first preincubated with 2 mm PLP and varying amounts of the inhibitor (PAG 0–4 mm) on ice for 30 min. This was followed by the addition of 5 mm l-cysteine and incubation at 37 °C for 30 min. The IC50 value was then estimated from a plot of percentage inhibition against PAG concentration. Isothermal Titration Calorimetry—hCSE protein was concentrated to 41 μm in 20 mm sodium phosphate, pH 7.8. PAG was prepared at 600 μm using the same buffer. PAG is loaded into the syringe and titrated against hCSE·PLP in a VP-ITC microcalorimeter (ITC 200; Microcal) performed under identical conditions. The dissociation constants were determined by least squares method, and the binding isotherm was fitted using Origin version 7.0 (Microcal), assuming a one-site binding model. Heat-released Q(i), from the ith injection is as follows. ΔQ(i)=Q(i)+dVVo(Q(i)+Q(i−1)2)−Q(i−1)(Eq. 3) All measurements were repeated at least twice for verification. Overall Structures—The structures of apo-hCSE, hCSE·PLP, and hCSE·PLP·PAG complexes were solved and refined to 2.4, 2.6, and 2.0 Å resolution, respectively (Table 1). The resolution of the hCSE·PLP·PAG complex significantly increased, most likely due to the improved quality and size of the crystal. hCSE exists entirely as a tetramer in the PLP-bound states (hCSE·PLP and hCSE·PLP·PAG complexes) and predominantly as tetramers in the apo state in solution as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation experiments (supplemental Fig. 1). We also report the observation of a tetramer in the asymmetric unit of the hCSE·PLP complex and hCSE·PLP·PAG complex structures. In the case of apo-hCSE, two dimers in the asymmetric unit form tetramers with symmetry-related dimers. Close contact between monomers of the tetramer are maintained by several hydrogen bonds and extensive hydrophobic interactions. Structures of the individual monomers of apo-hCSE, hCSE·PLP, and hCSE·PLP·PAG are very similar (root mean square deviation for apo-hCSE versus hCSE·PLP is 1.5 Å for 336 Cα atoms; apo-hCSE versus hCSE·PAG·PLP is 1.6 Å for 336 Cα atoms; hCSE·PLP versus hCSE·PLP·PAG is 0.3 Å for 390 Cα atoms) except for two loops near the active site region and a disordered loop at the monomer interface of the dimer in the apo-hCSE. Each hCSE monomer consists of two domains: 1) a larger PLP-binding domain (residues Ala9–His263) and 2) a smaller domain (residues Val264–Ser401) (Fig. 1a). The PLP binding domain consists of an α/β/α fold, a seven-stranded mixed β-sheet flanked on three sides by a total of eight α-helices. This central β-sheet consists of seven, mostly parallel, β-strands (β1↑β7↓β6↑β5↑β4↑β2↑β3↑) with only strand β7 being antiparallel to the rest. The small domain consists of a four-stranded antiparallel β-sheet (β8↓β9↑β11↓β10↑) and three helices on one side of the β-sheet. Sequence and Structural Similarity—CSE from various organisms forms a conserved group of PLP-dependent enzymes belonging to the family of cystathionine synthase-like proteins. In mammals, highest sequence similarities (80% sequence similarities among the 26 mammalian sequences compared) were obtained from CSE, hypothetical CSE, or CSE homologs. A search for structurally similar proteins was carried out with DALI (16Holm L. Park J. Bioinformatics (Oxford).. 2000; 16: 566-567Google Scholar) and BioXGEM (17Yang J.M. Tung C.H. Nucleic Acids Res... 2006; 34: 3646-3659Google Scholar, 18Tung C.H. Huang J.W. Yang J.M. Genome Biol... 2007; 8: R31Google Scholar). Most of the structural similarity was found around the active site region of CSE and the corresponding region in several PLP-dependent enzymes (supplemental Fig. 2). Active Site—The PLP binding site of hCSE is formed mainly by residues from the central seven-stranded β-sheet as well as α-helices that pack against one face of this sheet (Fig. 1a). PLP is anchored by strong hydrogen bonding between the phosphate moiety and residues contributed by neighboring subunits A (Gly90, Leu91, Ser209, and Thr211) and B (Tyr60 and Arg62) (supplemental Fig. 3). In addition, there are two other hydrogen bonding interactions (by Asp187 and Lys212) with PLP. Lys212 binds PLP, and Tyr114 exhibits aromatic π-stacking interactions to the pyridine ring of PLP (red color in Fig. 1, b and c). Such interactions have also been reported in a number of other PLP enzyme complexes. Fig. 1d shows the omit maps of PLP from the hCSE·PLP structure. Several CSE mutations are associated with cystathioninuria, two of which (T67I and Q240E) have recently been characterized (6Zhu W. Lin A. Banerjee R. Biochemistry.. 2008; 47: 6226-6232Google Scholar). Both mutants have been reported to have greatly reduced enzymatic activities relative to the wild-type enzyme. This reduction in activity has been attributed to lower affinity of PLP with the mutant enzymes. However, in our structure, the importance of these residues in PLP binding is not readily apparent, since they are not directly in contact with PLP. Open and Closed Conformations of the Active Site—When the structures of apo-hCSE and the hCSE·PLP/hCSE·PLP·PAG complex are superimposed, we observed that the active site is significantly open in the apoenzyme (Fig. 1, e and f). Detailed comparisons of structures of apo-hCSE with hCSE·PLP, hCSE·PLP·PAG, and other PLP-dependent enzymes reveal the magnitude of conformational changes between PLP-bound and apo forms of the enzyme. Distances between the equivalent atoms after superimposition of the molecules show that significant differences occur within the two loop regions Met110–Asn118 and Thr210–Met216. Both loops form walls on either side of the PLP binding cleft. When PLP is bound, these two loops fold back on the cofactor, thereby establishing effective interactions. This motion shifts the main chain of the central part of both loops at Tyr114 and Lys212 by as much as 8.6 and 7.0 Å, respectively. The largest displacements are those of the side chains of Tyr114 and Lys212, the tips of which move over 19.3 and 10.6 Å, respectively, with a simultaneous large movement of nearby side chains (supplemental movie 1). In the hCSE·PLP complex, there is a two-turn helix (residues Gly115–Tyr120) near Tyr114 that unwinds and becomes a loop in the apo-hCSE (Fig. 1b). The distance between the two loops in the open form is 22.7 Å, whereas the same distance in the closed form is 12.7 Å. In the hCSE·PLP complex, Lys212 is covalently bonded to the PLP cofactor through the formation of a Schiff base between the carbonyl group of PLP and the amino group of the Lys212 side chain (Fig. 1c). However, in the apo structure, Lys212 was found to have moved away from the PLP binding site. In the superimposed model of the structures, the distance between PLP of the hCSE·PLP complex and Lys212 of the apo structure is too large (11 Å apart) for nucleophilic attack and subsequent formation of the Schiff base. Previously, crystal structures of another PLP-dependent enzyme, namely threonine synthase from A. thaliana, were reported in the PLP-bound and PLP-free states, but no main chain conformational changes were observed in the active site region (19Thomazeau K. Curien G. Dumas R. Biou V. Protein Sci... 2001; 10: 638-648Google Scholar, 20Mas-Droux C. Biou V. Dumas R. J. Biol. Chem... 2006; 281: 5188-5196Google Scholar). Similarly, in the hCSE·PLP complex, one of the four monomers of the asymmetric unit is found in the closed conformation without PLP. This indicates that the PLP-free enzyme is highly dynamic and exists as an ensemble of multiple conformational states. In the hCSE·PLP structure, the N-terminal domain extends to the adjacent monomer and forms part of the neighboring PLP binding site. Residues Tyr60 and Arg62 electrostatically interact with the phosphate group of PLP (supplemental Fig. 3). However, in the apo-hCSE structure, the electron density map corresponding to this region (Asp28–Ser63) is disordered and indicates the flexibility of this segment due to the absence of PLP. Another missing region in apo-hCSE is from Thr355 to Val366; in the hCSE·PLP complex, this region does not directly interact with PLP, but it could stabilize one side of the PLP binding pocket. In the apo-hCSE, the Met110–Asn118 loop region occupies the Thr355–Val366 region of the hCSE·PLP complex. This is one possible reason why Thr355–Val366 of apo-hCSE is shifted further away from the PLP binding site and hence is disordered. l-cysteine needs to bind with PLP to eliminate the β-sulfhydryl group to produce H2S; therefore, without PLP, hCSE is unable to produce H2S. Oligomeric States of hCSE—In order to analyze the oligomeric states of apo- and PLP-bound hCSE, we performed size exclusion chromatography and analytical ultracentrifugation experiments. To best mimic the apo and PLP complex, two buffer conditions were chosen. In buffer A (10 mm sodium phosphate, pH 7.8), which favors the PLP-bound state, hCSE eluted predominantly as a tetramer. In contrast, hCSE exists as a mixture of tetramer and monomeric states in buffer B (50 mm sodium citrate, pH 4.0, and 10 mm l-cysteine), which favored the apo state of hCSE. This suggests that pH 4.0 and the presence of l-cysteine weakens interactions between ind" @default.
W2053104328 created "2016-06-24" @default.
W2053104328 creator A5002942595 @default.
W2053104328 creator A5012413850 @default.
W2053104328 creator A5018683664 @default.
W2053104328 creator A5018936281 @default.
W2053104328 creator A5021801679 @default.
W2053104328 creator A5028665730 @default.
W2053104328 creator A5035611310 @default.
W2053104328 creator A5038352899 @default.
W2053104328 creator A5073872551 @default.
W2053104328 creator A5079845863 @default.
W2053104328 creator A5081786054 @default.
W2053104328 date "2009-01-01" @default.
W2053104328 modified "2023-10-07" @default.
W2053104328 title "Structural Basis for the Inhibition Mechanism of Human Cystathionine γ-Lyase, an Enzyme Responsible for the Production of H2S" @default.
W2053104328 cites W1488975476 @default.
W2053104328 cites W1764016564 @default.
W2053104328 cites W1971142629 @default.
W2053104328 cites W1975317507 @default.
W2053104328 cites W1977373788 @default.
W2053104328 cites W1995017064 @default.
W2053104328 cites W2003531759 @default.
W2053104328 cites W2008664596 @default.
W2053104328 cites W2015407072 @default.
W2053104328 cites W2032560226 @default.
W2053104328 cites W2038840577 @default.
W2053104328 cites W2049540991 @default.
W2053104328 cites W2053673527 @default.
W2053104328 cites W2054064167 @default.
W2053104328 cites W2054506138 @default.
W2053104328 cites W2076451815 @default.
W2053104328 cites W2076949792 @default.
W2053104328 cites W2080861994 @default.
W2053104328 cites W2095151229 @default.
W2053104328 cites W2098807209 @default.
W2053104328 cites W2100126267 @default.
W2053104328 cites W2102574135 @default.
W2053104328 cites W2106315897 @default.
W2053104328 cites W2110397834 @default.
W2053104328 cites W2118972497 @default.
W2053104328 cites W2122698700 @default.
W2053104328 cites W2133442550 @default.
W2053104328 cites W2144081223 @default.
W2053104328 cites W2144390151 @default.
W2053104328 cites W255996521 @default.
W2053104328 doi "https://doi.org/10.1074/jbc.m805459200" @default.
W2053104328 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19019829" @default.
W2053104328 hasPublicationYear "2009" @default.
W2053104328 type Work @default.
W2053104328 sameAs 2053104328 @default.
W2053104328 citedByCount "159" @default.
W2053104328 countsByYear W20531043282012 @default.
W2053104328 countsByYear W20531043282013 @default.
W2053104328 countsByYear W20531043282014 @default.
W2053104328 countsByYear W20531043282015 @default.
W2053104328 countsByYear W20531043282016 @default.
W2053104328 countsByYear W20531043282017 @default.
W2053104328 countsByYear W20531043282018 @default.
W2053104328 countsByYear W20531043282019 @default.
W2053104328 countsByYear W20531043282020 @default.
W2053104328 countsByYear W20531043282021 @default.
W2053104328 countsByYear W20531043282022 @default.
W2053104328 countsByYear W20531043282023 @default.
W2053104328 crossrefType "journal-article" @default.
W2053104328 hasAuthorship W2053104328A5002942595 @default.
W2053104328 hasAuthorship W2053104328A5012413850 @default.
W2053104328 hasAuthorship W2053104328A5018683664 @default.
W2053104328 hasAuthorship W2053104328A5018936281 @default.
W2053104328 hasAuthorship W2053104328A5021801679 @default.
W2053104328 hasAuthorship W2053104328A5028665730 @default.
W2053104328 hasAuthorship W2053104328A5035611310 @default.
W2053104328 hasAuthorship W2053104328A5038352899 @default.
W2053104328 hasAuthorship W2053104328A5073872551 @default.
W2053104328 hasAuthorship W2053104328A5079845863 @default.
W2053104328 hasAuthorship W2053104328A5081786054 @default.
W2053104328 hasConcept C111472728 @default.
W2053104328 hasConcept C138885662 @default.
W2053104328 hasConcept C16525657 @default.
W2053104328 hasConcept C181199279 @default.
W2053104328 hasConcept C185592680 @default.
W2053104328 hasConcept C187566844 @default.
W2053104328 hasConcept C2777269803 @default.
W2053104328 hasConcept C2779201268 @default.
W2053104328 hasConcept C55493867 @default.
W2053104328 hasConcept C89611455 @default.
W2053104328 hasConceptScore W2053104328C111472728 @default.
W2053104328 hasConceptScore W2053104328C138885662 @default.
W2053104328 hasConceptScore W2053104328C16525657 @default.
W2053104328 hasConceptScore W2053104328C181199279 @default.
W2053104328 hasConceptScore W2053104328C185592680 @default.
W2053104328 hasConceptScore W2053104328C187566844 @default.
W2053104328 hasConceptScore W2053104328C2777269803 @default.
W2053104328 hasConceptScore W2053104328C2779201268 @default.
W2053104328 hasConceptScore W2053104328C55493867 @default.
W2053104328 hasConceptScore W2053104328C89611455 @default.
W2053104328 hasIssue "5" @default.
W2053104328 hasLocation W20531043281 @default.