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1.
Nat Immunol ; 18(8): 889-898, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28604720

RESUMO

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.


Assuntos
Complemento C1q/imunologia , Citotoxicidade Imunológica/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoterapia , Neoplasias/tratamento farmacológico , Fagocitose/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/imunologia , Linhagem Celular Tumoral , Cromatografia em Gel , Cromatografia Líquida , Complemento C1q/metabolismo , Cristalização , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Técnicas In Vitro , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/imunologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/imunologia , Espectrometria de Massas , Camundongos , Neoplasias/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptores de IgG/metabolismo , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem
3.
Proc Natl Acad Sci U S A ; 117(23): 13000-13011, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434918

RESUMO

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.


Assuntos
Cistationina gama-Liase/farmacologia , Metionina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cistationina gama-Liase/genética , Cistationina gama-Liase/isolamento & purificação , Cistationina gama-Liase/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Ensaios Enzimáticos , Humanos , Masculino , Metionina/sangue , Metionina/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/sangue , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Testes de Toxicidade Aguda , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nature ; 527(7579): 539-543, 2015 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-26524521

RESUMO

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.


Assuntos
Aspergillus fumigatus/enzimologia , Biocatálise , Ácidos Cetoglutáricos/metabolismo , Endoperóxidos de Prostaglandina/biossíntese , Sítios de Ligação , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Heme , Hidroxilação , Indóis/metabolismo , Ferro/metabolismo , Oxigênio/metabolismo , Tirosina/metabolismo
6.
Biochemistry ; 56(6): 876-885, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28106980

RESUMO

Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing ß- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.


Assuntos
Liases de Carbono-Enxofre/metabolismo , Cistationina gama-Liase/metabolismo , Metionina/metabolismo , Modelos Moleculares , Selenometionina/metabolismo , Substituição de Aminoácidos , Arginina/química , Biocatálise , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Domínio Catalítico , Cistationina/metabolismo , Cistationina gama-Liase/química , Cistationina gama-Liase/genética , Cisteína/metabolismo , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , Hidrólise , Mutagênese Sítio-Dirigida , Concentração Osmolar , Conformação Proteica , Engenharia de Proteínas , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
7.
ACS Catal ; 13(23): 15417-15426, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38058600

RESUMO

Ovothiol A and ergothioneine are thiol-histidine derivatives with sulfur substitutions at the δ-carbon or ε-carbon of the l-histidine imidazole ring, respectively. Both ovothiol A and ergothioneine have protective effects on many aging-related diseases, and the sulfur substitution plays a key role in determining their chemical and biological properties, while factors governing sulfur incorporation regioselectivities in ovothiol and ergothioneine biosynthesis in the corresponding enzymes (OvoA, Egt1, or EgtB) are not yet known. In this study, we have successfully obtained the first OvoA crystal structure, which provides critical information to explain their C-S bond formation regioselectivity. Furthermore, OvoATh2 exhibits several additional activities: (1) ergothioneine sulfoxide synthase activity akin to Egt1 in ergothioneine biosynthesis; (2) cysteine dioxygenase activity using l-cysteine and l-histidine analogues as substrates; (3) cysteine dioxygenase activity upon mutation of an active site tyrosine residue (Y406). The structural insights and diverse chemistries demonstrated by OvoATh2 pave the way for future comprehensive structure-function correlation studies.

8.
JACS Au ; 2(7): 1686-1698, 2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35911443

RESUMO

FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure-function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B (7) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•CoII•αKG•7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•FeII•αKG binary complex. Verruculogen (2) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.

9.
Biochemistry ; 50(35): 7600-11, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21809870

RESUMO

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.


Assuntos
Proteínas de Bactérias/química , Vias Biossintéticas/fisiologia , Isomerases/química , Homologia Estrutural de Proteína , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/fisiologia , Benzodiazepinonas/síntese química , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Isomerases/biossíntese , Isomerases/fisiologia , Cinética , Estrutura Terciária de Proteína/fisiologia , Pseudomonas putida/enzimologia , Transdução de Sinais/fisiologia , Staphylococcus aureus/enzimologia , Especificidade por Substrato/fisiologia
10.
FASEB J ; 24(1): 242-52, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19783784

RESUMO

Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 A, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widely than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase.


Assuntos
Fosfotransferases (Aceptor do Grupo Nitrogenado)/química , Fosfotransferases (Aceptor do Grupo Nitrogenado)/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Animais , Arginina Quinase/química , Arginina Quinase/genética , Arginina Quinase/metabolismo , Domínio Catalítico/genética , Creatina Quinase/química , Creatina Quinase/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Técnicas In Vitro , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Fosfotransferases (Aceptor do Grupo Nitrogenado)/genética , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pepinos-do-Mar/enzimologia , Pepinos-do-Mar/genética , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
11.
Cell Rep ; 32(3): 107909, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32697994

RESUMO

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Oncogenes , Proteínas Proto-Oncogênicas p21(ras)/química , Sequência de Aminoácidos , Manchas Café com Leite/genética , Domínio Catalítico , Análise Mutacional de DNA , Fator de Crescimento Epidérmico/farmacologia , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Células K562 , Neurofibromatose 1/genética , Neurofibromina 1/genética , Fosforilação , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
12.
Sci Rep ; 9(1): 10512, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324887

RESUMO

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.


Assuntos
Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/química , Acetilação , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Humanos , Ligação de Hidrogênio , Magnésio/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
13.
Cell Chem Biol ; 25(5): 519-529.e4, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29503207

RESUMO

Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.


Assuntos
Ergotioneína/metabolismo , Proteínas Fúngicas/metabolismo , Liases/metabolismo , Neurospora crassa/metabolismo , Vias Biossintéticas , Domínio Catalítico , Cristalografia por Raios X , Proteínas Fúngicas/química , Liases/química , Neurospora crassa/química , Conformação Proteica , Fosfato de Piridoxal/metabolismo , Especificidade por Substrato , Ácidos Sulfênicos/metabolismo
14.
Nat Med ; 23(1): 120-127, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869804

RESUMO

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.


Assuntos
Neoplasias da Mama/metabolismo , Cistationina gama-Liase/farmacologia , Cisteína/efeitos dos fármacos , Cistina/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/metabolismo , Polietilenoglicóis/farmacologia , Neoplasias da Próstata/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cisteína/metabolismo , Cistina/metabolismo , Feminino , Glutationa/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transplante de Neoplasias , Estresse Oxidativo , Proteína Supressora de Tumor p53/genética
15.
ACS Chem Biol ; 8(9): 2042-52, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23844594

RESUMO

The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats, through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intramolecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 does not dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 toward the CTD peptide by 4-fold. To our surprise, even though the intramolecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas de Drosophila/química , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Proteínas Tirosina Fosfatases/química , RNA Polimerase II/química , Fatores de Poliadenilação e Clivagem de mRNA/química
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