LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.
Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.
A member of the novel protein kinase C (PKC) subfamily, PKCθ, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKCθ in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKCθ at 2.0-Å resolution. Human recombinant PKCθ kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKCθ autocatalytic phosphorylation and activation during bacterial expression. The PKCθ-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKCθ selective leads. A member of the novel protein kinase C (PKC) subfamily, PKCθ, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKCθ in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKCθ at 2.0-Å resolution. Human recombinant PKCθ kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKCθ autocatalytic phosphorylation and activation during bacterial expression. The PKCθ-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKCθ selective leads. Structure of a Novel PKC Family MemberJournal of Biological ChemistryVol. 279Issue 48PreviewZhang-Bao Xu and colleagues have determined the crystal structure of a protein kinase C (PKC) isozyme, in this case the novel PKC family member PKCθ. This structure should prove extremely useful in the rational design of small molecule inhibitors of PKCθ, which has been implicated in T-cell-mediated disease processes including inflammation and autoimmunity. Full-Text PDF Open Access Inhibitors of PKC 1The abbreviations used are: PKC, PKA, and PKB, protein kinase C, A, and B, respectively; PKI, protein kinase inhibitor; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate); DTT, dithiothreitol; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PDB, Protein Data Bank; N- and C-lobe, N- and C-terminal lobe, respectively; HM, hydrophobic motif. are currently being used in clinical trials for various types of cancer, and a PKCβ inhibitor is being used in clinical trials for diabetes-related retinopathy (1Cohen P. Nat. Rev. Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1869) Google Scholar). PKC and PKB/AKT kinase domains are related by sequence homology; however, there are key structural differences in the regulatory domains and second messenger cofactor requirements. PKB/AKT contains an N-terminal pleckstrin homology domain regulated by phosphoinositide second messengers, a central catalytic kinase domain, and a C-terminal regulatory region facilitating key protein-protein interactions with signaling molecules like Src kinase (2Jiang T. Qiu Y. J. Biol. Chem. 2003; 278: 15789-15793Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). PKC kinases can be regulated by calcium, diacylglycerol, and phorbol esters and are divided into three subfamilies based on their cofactor requirements (3Tan S.L. Parker P.J. Biochem. J. 2003; 376: 545-552Crossref PubMed Scopus (212) Google Scholar): conventional (PKCα, PKCβI, PKC βII, PKCγ), novel (PKCδ, PKCϵ, PKCθ, PKCη), and atypical (PKCζ, PKCι, PKCλ, PKCμ) isoforms. PKC kinases have a C-terminal catalytic kinase domain and an N-terminal regulatory co-factor-binding domain. The N-terminal motifs comprise the phosphatidylserine- and diacylglycerol-binding C1 motifs and calcium-binding C2 domain, in addition to a pseudosubstrate sequence motif that is regulated by cofactor binding. The closely related PKC isoforms have been shown to have important roles in T cells (PKCα, PKCθ), B cells and mast cells (PKCβ, PKCδ), and macrophages (PKCϵ), contributing to adaptive and innate immunity (3Tan S.L. Parker P.J. Biochem. J. 2003; 376: 545-552Crossref PubMed Scopus (212) Google Scholar). Both PKCθ and PKB/AKT are implicated in T cell signaling leading to T cell activation and survival (4Arendt C.W. Albrecht B. Soos T.J. Littman D.R. Curr. Opin. Immunol. 2002; 14: 323-330Crossref PubMed Scopus (93) Google Scholar, 5Bauer B. Krumbock N. Fresser F. Hochholdinger F. Spitaler M. Simm A. Uberall F. Schraven B. Baier G. J. Biol. Chem. 2001; 276: 31627-31634Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 6Bauer B. Krumbock N. Ghaffari-Tabrizi N. Kampfer S. Villunger A. Wilda M. Hameister H. Utermann G. Leitges M. Uberall F. Baier G. Eur. J. Immunol. 2000; 30: 3645-3654Crossref PubMed Scopus (48) Google Scholar). However, the expression and role of PKCθ are relatively restricted to T cells, with signaling in response to TCR stimulation contributing to T cell activation and cytokine production (7Altman A. Villalba M. Immunol. Rev. 2003; 192: 53-63Crossref PubMed Scopus (98) Google Scholar, 8Pfeifhofer C. Kofler K. Gruber T. Tabrizi N.G. Lutz C. Maly K. Leitges M. Baier G. J. Exp. Med. 2003; 197: 1525-1535Crossref PubMed Scopus (288) Google Scholar, 9Sun Z. Arendt C.W. Ellmeier W. Schaeffer E.M. Sunshine M.J. Gandhi L. Annes J. Petrzilka D. Kupfer A. Schwartzberg P.L. Littman D.R. Nature. 2000; 404: 402-407Crossref PubMed Scopus (792) Google Scholar). PKCθ co-localizes to the immunological synapse in response to T cell activation (10Diaz-Flores E. Siliceo M. Martinez A.C. Merida I. J. Biol. Chem. 2003; 278: 29208-29215Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Thus, PKCθ inhibition is potentially desirable in T cell leukemias (11Villalba M. Altman A. Curr. Cancer Drug Targets. 2002; 2: 125-137Crossref PubMed Scopus (34) Google Scholar) and T cell-mediated allergic and autoimmune disorders. Among AGC superfamily kinases, the kinase domain crystal structures have been determined for both PKB/AKT- and cAMP-dependent PKA (12Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1370) Google Scholar) but not for a PKC isoform. The homologies in the kinase domain ATP-binding site have been a challenge in the development of highly specific inhibitors as disease therapies (1Cohen P. Nat. Rev. Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1869) Google Scholar, 13Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1467) Google Scholar). Structural elucidation of kinase active sites and comparison with that of closely related family members greatly increases our understanding of the mechanism of enzyme action and divulges issues regarding selectivity. A Rho kinase (AGC superfamily) inhibitor Fasudil/HA1077/1-(5-isoquinolinesulphonyl)homopiperazine HCl, belonging to the isoquinoline sulfonamide class of compounds, also inhibits both PKA and PKC in a reversible and ATP competitive manner (14Hidaka H. Inagaki M. Kawamoto S. Sasaki Y. Biochemistry. 1984; 23: 5036-5041Crossref PubMed Scopus (2329) Google Scholar). This kinase inhibitor is a therapeutic drug in treating cerebral vasospasm and has recently been co-crystallized with the PKA catalytic subunit to define key interactions of the kinase inhibitor within the ATP binding site (15Breitenlechner C. Gassel M. Hidaka H. Kinzel V. Huber R. Engh R.A. Bossemeyer D. Structure (Lond.). 2003; 11: 1595-1607Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The crystal structure of PKA revealed that the invariant amino acids in the highly conserved kinase catalytic core are clustered at the sites of nucleotide binding and catalysis (13Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1467) Google Scholar). The PKB/AKT active enzyme structure complexed with AMP-PNP and substrate peptide revealed mechanistic implications of key phosphorylations of the kinase domain (16Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (436) Google Scholar). More recently, the comparison of the PKB/AKT structure with PKA structure (17Gassel M. Breitenlechner C.B. Ruger P. Jucknischke U. Schneider T. Huber R. Bossemeyer D. Engh R.A. J. Mol. Biol. 2003; 329: 1021-1034Crossref PubMed Scopus (48) Google Scholar) provided explanations for distinct substrate specificities of the similar active kinase domain conformations. Yang et al. (16Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (436) Google Scholar) have also speculated potential PKC, SGK, p70, and p90 S6 kinase substrate interactions based on homologies with the GSK-3 substrate binding residues of PKB/AKT. Till now these mechanistic insights remained to be confirmed by a PKC crystal structure, and the studies presented here attempt to define PKC kinase domain characteristics. We report the x-ray structure elucidation of the PKCθ catalytic domain, with mechanistic insights into similarities and distinctions from the closely related PKB/AKT and PKA catalytic domains. In addition, the structural information of the staurosporine-complexed PKCθ kinase domain presented here will aid in the rational design and optimization of selective small molecule inhibitors for therapeutic use for inhibiting PKCθ in specific targeting of T cells. Expression and Purification—The C-terminal catalytic domain of PKCθ (residues 362–706) was cloned into a pET-16b expression vector. This vector introduced a hexahistidine tag to the C terminus of the expressed protein, and a methionine-glycine amino acid pair was introduced at the N terminus by cloning. The plasmid was used to transform Escherichia coli strain BL21-DE3 for overexpression. A 10-liter cell culture was expanded at 37 °C to an A600 of about 0.4. The temperature was then lowered to 25 °C before addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.1 mm to induce expression. The cells were grown for an additional 4 h before they were harvested. Harvested cells were resuspended in 25 mm Tris, pH 8.0, 25 mm NaCl, 5 mm 2-mercaptoethanol, 5 mm imidazole, 50 μm ATP, and protease inhibitors and lysed using a microfluidizer. The lysate was applied to 20 ml of nickel-nitrilotriacetic acid resin for 1 h at 4 °C. The resin was subsequently poured as a chromatography column and washed extensively with the same buffer including 25 mm imidazole. Protein bound to the resin was eluted with 200 mm imidazole buffer and then immediately loaded onto an anion exchanger HQ. The column was washed with 25 mm Tris, pH 8.0, 25 mm NaCl, 5 mm DTT, 50 μm ATP before being resolved by the application of a linear gradient from 25 to 500 mm NaCl. Fractions containing PKCθ were selected by SDS-PAGE, pooled, and diluted 2-fold with 25 mm Tris, pH 8.0, 5 mm DTT and loaded onto a heparin chromatography column. The flow-through was applied to a hydroxyapatite column and washed extensively with 25 mm Tris, pH 8.0, 50 mm NaCl, 5 mm DTT. A linear gradient of sodium phosphate from 0 to 100 mm eluted the target protein. The protein was then sized as a monomer on a Superdex 200 size exclusion chromatography column. The purified protein was dialyzed overnight against 25 mm Tris, pH 8.0, 50 mm NaCl, 5 mm DTT and concentrated to 7 mg/ml (determined by the Bradford assay) before being used for crystallization experiments. Kinase Assays and Data Analysis—ATP, ADP, phosphoenolpyruvate, NADH, pyruvate kinase/lactate dehydrogenase enzymes, staurosporine, acetonitrile, and the buffer HEPES were purchased from Sigma. Peptide substrate was purchased from AnaSpec (San Jose, CA), Syn Pep (Dublin, CA), or Open Biosystems (Huntsville, AL). The enzymatic activity determined using the coupled pyruvate kinase lactate dehydrogenase assay followed spectrophotometrically at 340 nm. The standard reaction, except where indicated, was carried out in 25 mm HEPES, pH 7.5, 10 mm MgCl2, and 2 mm DTT, 0.008% Triton, 100 mm NaCl, 20 units of pyruvate kinase, 30 units of lactate dehydrogenase, 0.25 mm NADH, 2 mm phosphoenolpyruvate. The PKCθ concentration was in the range of 0.156–0.312 μg/ml. The kinetic analysis was carried out in a 384-well plate at 25 °C on a Molecular Devices spectrophotometer in a final volume of 0.080 ml. The steady state kinetic parameters were determined in the buffer described above containing varied sucrose or Ficoll 400. Data were fitted to Equation 1 for normal Michaelis-Menten kinetics,ν=Vmax[S]/Km+[S](Eq.1) where [S] is the substrate, Vmax is the maximum enzyme velocity, and Km is the Michaelis constant. For inhibition kinetics the data were fitted to a competitive inhibition model in Equation 2,ν=Vmax[S]/Km(1+[I]/Kis)+[S](Eq.2) where Kis is the slope inhibition constant. The data were analyzed using Sigma Plot 2000 Enzyme Kinetics Module from SPSS Science (Richmond, CA). Crystallization and Structure Determination—Crystals with staurosporine were obtained at 18 °C from hanging drops containing 1 μl of protein:staurosporine solution (at ∼1:1 molar ratio) and 1 μl of precipitating solution (2 m ammonium sulfate, 40 mm DTT, and 0.1 m bis-tris, pH 5.0). The crystals belong to the monoclinic space group C2, with one protein-staurosporine complex in crystallographic asymmetric unit. Prior to data collection, crystals were stabilized in solution containing mother liquor plus 25% glycerol and flash-frozen in a 100 K nitrogen stream. The x-ray diffraction data were collected to 2-Å resolution at the Advanced Light Source (Berkeley, CA) using a Quantum-4 CCD detector (Area Detector Systems) and then reduced and scaled with HKL2000 (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The structure was solved by molecular replacement with AMORE (19P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) using the structure of PKA (PDB code: 1STC (20Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure (Lond.). 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar)), in which the A-helix, glycine-rich loop, activation loop, and C-terminal tail were omitted from the search model. The rotation and translation function solutions were found using data from 8 to 3.5 Å. The BUSTER program (21Bricogne G. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 37-60Crossref PubMed Google Scholar) with TNT (22Tronrud D.E. Methods Enzymol. 1997; 277: 306-319Crossref PubMed Scopus (127) Google Scholar) were applied in generating maximum entropy omit maps to overcome model bias and to produce a more detailed map for the bound inhibitor. Several rounds of rebuilding (QUANTA, Molecular Simulations, Inc.) and refinement were performed. To further reduce model bias and to generate better maps, an "average map" was calculated using CNS (23Brunger A.T. Adams P.D. Clore G.M. De Lano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuzewski J. Nigles M. Pannu N.S. Read R.J. Rice L.M. Somonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) by overlapping seven protein kinase coordinates including those of PKA. The resulting electron density maps were of better quality, especially for loop regions in the N-terminal lobe (N-lobe). The model was further rebuilt and refined, and the quality of the model was judged by the decrease in R-factors. Refinement converged after many rebuilding cycles to an R-factor of 0.201 and Rfree of 0.216. Crystallographic data collection and refinement statistics are summarized in Table I. The final model contains protein residues 377–649 and 688–696, two phosphate groups attached at Thr-538 and Ser-695, one staurosporine molecule, and 115 water molecules. Residues 362–376 from the N terminus, C-terminal region 650–687, and residues 697–706 at the very C terminus were not detected in the electron density maps due to disordering. Structural figures were generated using PyMOL (24De Lano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) and QUANTA (Molecular Simulations, Inc.).Table IStatistics for data collection and refinementData collectionSpace groupC2Unit cell dimensions (Å)a = 139.6; b = 42.4; c = 67.7; β = 116.2°Resolution range (Å)20.0-2.0Completeness (%)94.7Unique reflections23,134 (1,520)Average I/σ(I)19.3 (2.5)Rsym (%)aRsym = Σ|Ih - 〈Ih〉|/Σ Ih, where 〈Ih〉 is the average intensity over symmetry equivalents. Numbers in parentheses reflect statistics for the last resolution shell (2.07-2.0 Å)4.1 (23.6)Model refinementMaximum resolution (Å)2.0Number of reflections (free)21,992 (818)Rwork/RfreebRwork = Σ||Fobs - |Fcalc|/Σ|Fobs|, where Rfree is equivalent to Rwork but calculated for a randomly chosen 4% of reflections omitted from the refinement process20.1/21.6No. of protein atoms2,353No. of waters115Root mean square deviationsBonds (Å)0.005Angles (°)1.082a Rsym = Σ|Ih - 〈Ih〉|/Σ Ih, where 〈Ih〉 is the average intensity over symmetry equivalents. Numbers in parentheses reflect statistics for the last resolution shell (2.07-2.0 Å)b Rwork = Σ||Fobs - |Fcalc|/Σ|Fobs|, where Rfree is equivalent to Rwork but calculated for a randomly chosen 4% of reflections omitted from the refinement process Open table in a new tab Modeling of Peptide Substrates Bound to PKCθ—The PKCθ-staurosporine complex structure was aligned to a structure of PKB-β (AKT-2) in complex with a GSK-3 peptide (16Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (436) Google Scholar), PDB code: 106L) via automatic alignment (using weights of 1.0 for both sequence homology and structural homology) in the Protein Design module of Quanta (Accelrys (2004), San Deigo, CA). The alignment was further adjusted manually to improve the overlap of the "hinge region" backbone (residues 459–461 in PKCθ) between the two structures. The initial positioning in the PKCθ structure of the ATP analog AMP PNP in the ATP binding site, the peptide in its binding site, and the glycine-rich loop was determined by the alignment. The GSK-3 peptide was mutated to the PKCθ activation segment (532GDAKTNTFCG541) peptide in one case and the hydrophobic motif peptide (689NMFRNFSFMN698) in the other case. The conformation and position of residues 386–395 (the glycine-rich loop plus two residues on either side) was taken from the PKB-β structure. First, the attachment points for the loop, residues 386–388 and 696–698, were energy minimized keeping the remainder of the structure fixed. Next, the peptide, the glycine-rich loop, AMP-PNP, and the surrounding residues (with an atom within 8 Å) were minimized subject to decreasing harmonic constraints. Finally, the peptide was fully minimized keeping the remainder of the structure fixed. The minimizations were carried out using CHARMm (Ref. 25Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14019) Google Scholar; Accelrys (2004), San Deigo, CA) with the all atom force field (26MacKerell A.D. Bashford D. Bellott M. Dunbrack R.L. Evanseck J.D. Field M.J. Fischer S. Gao J. Guo H. Ha S. Joseph-McCarthy D. Kuchnir L. Kuczera K. Lau F.T.K. Mattos C. Michnick S. Ngo T. Nguyen D.T. Prodhom B. Reiher W.E. Roux B. Schlenkrich M. Smith J.C. Stote R. Straub J. Watanabe M. Wiorkiewicz-Kuczera J. Yin D. Karplus M. J. Phys. Chem. B. 1998; 102: 3586-3616Crossref PubMed Scopus (11819) Google Scholar). Merck Molecular Force Field 94s (27Halgren T.A. J. Comput. Chem. 1996; 17: 490-519Crossref Scopus (4384) Google Scholar) charges were used for AMP-PNP. Analysis of Protein Construct and Overall Structure—The bacterially expressed PKCθ kinase domain used for structure determination (residues 362–706) showed higher molecular weight than expected. Treatment by λ-phosphatase and subsequent molecular mass determination by electrospray ionization-mass spectrometry indicated that the protein is phosphorylated at either six or seven amino acid residues (roughly a 50:50 mixture). The purified PKCθ kinase domain was shown to have a higher specific activity than the full-length, commercially available PKCθ. Further characterizations of its catalytic activity demonstrated that it has an apparent Km of ∼49 μm for ATP and Km of ∼6.5 μm for a peptide derived from the PKCα pseudosubstrate, as indicated in Table II. Details of mass spectrometric analysis of phosphorylation sites and catalytic domain enzymatic characterization will be published elsewhere. 2Z.-B. Xu, D. Chaudhary, S. Olland, S. Wolfrom, R. Czerwinski, K. Malakian, L. Lin, M. L. Stahl, D. Joseph-McCarthy, C. Benander, L. Fitz, R. Greco, W. S. Somers, and L. Mosyak, submitted for publication. Consistent with the above data and as discussed further below, the enzyme crystallized in its phosphorylated state and in an active conformation.Table IIKinetic parameters for substrates and inhibitors of PKCθSteady state kinetic parameters for PKCθVaried substrateKm(app)kcatkcat/KmμMS-1M-1 S-1FARKGSLRQ6.5 ± 0.818 ± 12,700,000ATP49 ± 518 ± 1360,000Inhibition constants for PKCθSubstrateInhibitorInhibition patternInhibition constantATPStaurosporineCompetitiveKis = 0.33 ± 0.04 nm Open table in a new tab As outlined in the Introduction, the kinase domains of PKA, PKB/AKT, and PKC are highly homologous. Within the PKC subfamily, isozymes display more than 60% sequence identity in the kinase domain and share three conserved phosphorylation motifs (Fig. 1). The overall fold of the catalytic domain of PKCθ is very close to other protein kinase structures solved, with most similarities to those of PKB/AKT and PKA (Fig. 2). The conserved core of the structure is made of a small N-terminal lobe (residues 377–461) and a large C-terminal lobe (C-lobe) (residues 466–696), connected by a hinge linker (residues 462–465). The N-lobe is based on a five-stranded β-sheet (β1–β5) and two α-helices (αB and αC), and the C-lobe is mostly helical consisting of eight α-helices (αD–αK). The ATP-binding site, occupied by staurosporine, with the adjacent peptide-substrate binding site open to solvent, constitute the active site cleft at the interface of the two lobes. The glycine-rich phosphate-binding loop (GXGXXG), which shows a broad range of conformations (12Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1370) Google Scholar), including multiple conformations observed within a single crystal (15Breitenlechner C. Gassel M. Hidaka H. Kinzel V. Huber R. Engh R.A. Bossemeyer D. Structure (Lond.). 2003; 11: 1595-1607Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), connects the β1 and β2 strands (residues 386–394) and adopts a fixed and closed conformation.Fig. 2Overall structure of the PKCθ-staurosporine complex and comparison with the structure of the PKA-staurosporine-PKI complex.A, ribbon representation of the PKCθ kinase domain structure. The N-lobe is cyan and the C-lobe is blue. The glycine-rich loop, activation loop, and HM segment are highlighted in red. Staurosporine and phosphorylated residues are shown in stick representation. B, superposition of PKCθ-staurosporine (color coding is the same as described for A) and PKA-staurosporine-PKI structures (yellow ribbons and sticks; PDB code: 1STC (20Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure (Lond.). 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar)). Phosphorylated sites in PKA are also shown. Structures were aligned using the central helices from the C-lobe. Both staurosporine-bound kinases display intermediate lobe structures with conformational differences in the glycine-rich loop.View Large Image Figure ViewerDownload (PPT) Catalytic key residues (Lys-409, Asp-504, and Asp-522), invariant in all protein kinases, preserve intramolecular interactions observed in active kinase structures, in accordance with the structural criteria used to define catalytically active kinase conformations (12Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1370) Google Scholar). As in most Ser/Thr kinase structures reflecting active enzymes (28Johnson L.N. Noble E.M. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar), helix αC (residues 421–437) is properly aligned for substrate binding and catalysis, and the activation loop (residues 522–544) bearing the essential phosphothreonine Thr-538(P) (29Liu Y. Graham C. Li A. Fisher R.J. Shaw S. Biochem. J. 2002; 361: 255-265Crossref PubMed Scopus (104) Google Scholar) is well ordered and in an extended conformation. The C-terminal hydrophobic motif (HM) FXXFS* (residues 691–695), another conserved feature across AGC family, is adjacent to the hydrophobic groove of the N-lobe, in a location similar to the FXXF-binding pocket in PKA and PKB. Like in PKB, but in contrast to PKA that terminates with the FXXF sequence, HM in PKCθ contains phosphoserine at position 695 (Ser-695(P)), a phosphorylation that substantially effects the PKCθ kinase activity (29Liu Y. Graham C. Li A. Fisher R.J. Shaw S. Biochem. J. 2002; 361: 255-265Crossref PubMed Scopus (104) Google Scholar). PKCθ has an additional conserved phosphorylation site referred to as the turn motif (residues 662–685 and Fig. 1). In the structure, a long polypeptide linker between the kinase domain and the C-terminal HM (residues 650–687) is disordered; therefore the region corresponding to the turn motif is not defined in the electron density maps. There is also no observable electron density for residues C-terminal to HM (residues 697–706). In both PKA and PKB, the corresponding C-terminal linker is structurally ordered extending across the ATP-binding cleft (Fig. 2B). Compared with PKB, where HM is contained in the 17-residue-long stretch, HM seen in the PKCθ structure is considerably shorter (residues 688–696) and displays high b-factor values. Together these observations indicate that the C-terminal tail of PKCθ encompassing the turn motif and HM is intrinsically flexible either in this particular crystal form or in the absence of other functional domains or substrates. Binding of Staurosporine—The natural broad-spectrum kinase inhibitor staurosporine, with micromolar potency against only few kinases and low nanomolar potency against most kinases, has been shown to have a higher degree of selectivity toward PKC kinases (as reviewed in Ref. 30Meggio F. Donella-Deana A. Ruzzene M. Brunati A.M. Cesaro L. Guerra B. Meyer T. Mett H. Fabbro D. Furet P. Dobrowska G. Pinna L.A. Eur. J. Biochem. 1995; 234: 317-322Crossref PubMed Scopus (251) Google Scholar). Our kinetics data on PKCθ activity in the presence of staurosporine indicate it to be a strong, ATP competitive inhibitor with a Ki value of 0.33 nm (Table II). Associations maintaining this tight interaction are clearly and well defined by electron density (Fig. 3A) and will be described in comparison with the binding of staurosporine to PKA (PDB code: 1STC (20Prade L. Engh R.A. Girod A. Kinzel V. Huber R. Bossemeyer D. Structure (Lond.). 1997; 5: 1627-1637Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar)). As expected, these high affinity ligands bind to the related enzymes in a generally similar binding mode. Staurosporine resides in the ATP-binding pocket, forming four potential hydrogen bonds with the protein backbone (Fig. 3A) and extensive van der Waals contacts with the surrounding residues from both lobes and the hinge linker (Table III). Hydrogen bonding between the lactam ring of staurosproine (N-1, O-5) and the hinge polypeptide (O-459, N-461) is well reproduced in all protein kinase-staurosporine structur
Type IIA topoisomerases both manage the topological state of chromosomal DNA and are the targets of a variety of clinical agents. Bisdioxopiperazines are anticancer agents that associate with ATP-bound eukaryotic topoisomerase II (topo II) and convert the enzyme into an inactive, salt-stable clamp around DNA. To better understand both topo II and bisdioxopiperazine function, we determined the structures of the adenosine 5′-[β,γ-imino]-triphosphate-bound yeast topo II ATPase region ( Sc T2-ATPase) alone and complexed with the bisdioxopiperazine ICRF-187. The drug-free form of the protein is similar in overall fold to the equivalent region of bacterial gyrase but unexpectedly displays significant conformational differences. The ternary drug-bound complex reveals that ICRF-187 acts by an unusual mechanism of inhibition in which the drug does not compete for the ATP-binding pocket, but bridges and stabilizes a transient dimer interface between two ATPase protomers. Our data explain why bisdioxopiperazines target ATP-bound topo II, provide a structural rationale for the effects of certain drug-resistance mutations, and point to regions of bisdioxopiperazines that might be modified to improve or alter drug specificity.
IL‐21 is a pleiotropic type I cytokine that is secreted by activated CD4 T cells. IL21 has been found to induce apoptosis in resting B cells but promotes class switch recombination and plasma cell differentiation of activated B cells. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a break down in both T and B cell tolerance, and the development of anti‐nuclear antibodies. Generation of high titers of IgG antibodies reactive with dsDNA coincides with disease progression. Thus, factors that promote B cell activation and antibody production, such as IL‐21, may be important for the development of SLE. We examined the role of IL‐21R in the cGVHD model of lupus‐like disease using IL‐21R deficient mice. Cells from bm12 mice were transferred into C57Bl/6 mice or into IL21R KO mice on the C57Bl/6 background to determine whether the lack of IL‐21R in the host would affect antibody production, immune complex deposition, and kidney pathology. All of these parameters were decreased in the IL21R KO recipient mice. We also show that treatment of MRL‐Faslpr mice with an antagonistic anti‐IL‐21R antibody significantly reduced anti‐dsDNA IgG serum antibody levels and prevented IC deposition and kidney pathology in these mice. Therefore, blockade of the IL‐21 pathway is a potential therapeutic target for the treatment of human lupus.
Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.
Protein kinase C θ (PKCθ), a member of the Ca2+-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKCθ-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKCθ is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKCθ autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKCθ kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKCθ kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCKθ362-706 as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATPγS on PKCθ catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKCθ. These results should aid the design and discovery of PKCθ antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.
While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with <2% high molecular weight species present after 7 weeks at 4 °C and viscosity <15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.
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