The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.
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
Abstract GDF15 is a distant TGF-β family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.
