RESUMO
A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
Assuntos
Trifosfato de Adenosina , Ligação Proteica , Ribonucleotídeo Redutases , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Anaerobiose , Nucleotídeos de Desoxiadenina/metabolismo , Domínio Catalítico , Conformação Proteica , Especificidade por Substrato , Multimerização Proteica , Modelos MolecularesRESUMO
Galectins play biological roles in immune regulation and tumor progression. Ligands with high affinity for the shallow, hydrophilic galectin-3 ligand binding site rely primarily on a galactose core with appended aryltriazole moieties, making hydrophobic interactions and π-stacking. We designed and synthesized phenyl sulfone, sulfoxide, and sulfide-triazolyl thiogalactoside derivatives to create affinity-enhancing hydrogen bonds, hydrophobic and π-interactions. Crystal structures and thermodynamic analyses revealed that the sulfoxide and sulfone ligands form hydrogen bonds while retaining π-interactions, resulting in improved affinities and unique binding poses. The sulfoxide, bearing one hydrogen bond acceptor, leads to an affinity decrease compared to the sulfide, whereas the corresponding sulfone forms three hydrogen bonds, two directly with Asn and Arg side chains and one water-mediated to an Asp side chain, respectively, which alters the complex structure and increases affinity. These findings highlight that the sulfur oxidation state influences both the interaction thermodynamics and structure.
Assuntos
Galectina 3 , Galectinas , Galectina 3/metabolismo , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Enxofre , Sulfetos , Sulfonas , SulfóxidosRESUMO
Interleukin-27 (IL-27) uniquely assembles p28 and EBI3 subunits to a heterodimeric cytokine that signals via IL-27Rα and gp130. To provide the structural framework for receptor activation by IL-27 and its emerging therapeutic targeting, we report here crystal structures of mouse IL-27 in complex with IL-27Rα and of human IL-27 in complex with SRF388, a monoclonal antibody undergoing clinical trials with oncology indications. One face of the helical p28 subunit interacts with EBI3, while the opposite face nestles into the interdomain elbow of IL-27Rα to juxtapose IL-27Rα to EBI3. This orients IL-27Rα for paired signaling with gp130, which only uses its immunoglobulin domain to bind to IL-27. Such a signaling complex is distinct from those mediated by IL-12 and IL-23. The SRF388 binding epitope on IL-27 overlaps with the IL-27Rα interaction site explaining its potent antagonistic properties. Collectively, our findings will facilitate the mechanistic interrogation, engineering, and therapeutic targeting of IL-27.
Assuntos
Interleucina-27 , Humanos , Camundongos , Animais , Receptor gp130 de Citocina/metabolismo , Receptores de Citocinas/metabolismo , Interleucina-12 , Citocinas , Anticorpos Monoclonais/farmacologia , Epitopos , Interleucina-23RESUMO
Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria - including well-known pathogens such as Mycobacterium tuberculosis - NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.
Assuntos
Ribonucleotídeo Redutases , Streptomyces coelicolor , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Nucleotídeos/química , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Streptomyces coelicolor/metabolismoRESUMO
Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.
Assuntos
Antígenos CD28 , Neoplasias , Animais , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linfócitos TRESUMO
We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Desenvolvimento de Medicamentos/métodos , Fragmentos Fab das Imunoglobulinas/farmacologia , Engenharia de Proteínas , Sequência de Aminoácidos/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/ultraestrutura , Becaplermina/antagonistas & inibidores , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Concentração Inibidora 50 , Injeções Intravítreas , Masculino , Modelos Moleculares , Estudo de Prova de Conceito , Conformação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
The galectins are a family of galactose-binding proteins playing key roles in inflammatory processes and cancer. However, they are structurally very closely related, and discovery of highly selective inhibitors is challenging. In this work, we report the design of novel inhibitors binding to a subsite unique to galectin-3, which confers both high selectivity and affinity towards galectin-3. Olefin cross metathesis between allyl ß-C-galactopyranosyl and 1-vinylnaphthalenes or acylation of aminomethyl ß-C-galactopyranosyl with 1-naphthoic acid derivatives gave C-galactopyranosyls carrying 1-naphthamide structural elements that interacted favorably with a galectin-3 unique subsite according to molecular modeling and X-ray structural analysis of two inhibitor-galectin-3 complexes. Affinities were down to sub-µM and selectivities over galectin-1, 2, 4 N-terminal domain, 4 C-terminal domain, 7, 8 N-terminal domain, 9 N-terminal domain, and 9 C-terminal domain were high. These results show that high affinity and selectivity for a single galectin can be achieved by targeting unique subsites, which holds promise for further development of small and selective galectin inhibitors.
Assuntos
Galactose , Galectina 3/química , Acilação , Proteínas Sanguíneas , Cristalografia por Raios X , Galactose/análogos & derivados , Galactose/síntese química , Galactose/química , Galectinas , Humanos , Domínios ProteicosRESUMO
Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.
Assuntos
Bacillus anthracis/enzimologia , Bacillus anthracis/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Metaloproteases/metabolismo , Cristalografia por Raios X , FMN Redutase/química , FMN Redutase/genética , FMN Redutase/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Mononucleotídeo de Flavina/metabolismo , Metaloproteases/química , Metaloproteases/genética , Ribonucleotídeo RedutasesRESUMO
Cathepsin C (CatC) is a dipeptidyl-exopeptidase which activates neutrophil serine protease precursors (elastase, proteinase 3, cathepsin G and NSP4) by removing their N-terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil differentiation. The resulting active proteases are implicated in chronic inflammatory and autoimmune diseases. Hence, inhibition of CatC represents a therapeutic strategy to suppress excessive protease activities in various neutrophil mediated diseases. We designed and synthesized a series of dipeptidyl cyclopropyl nitrile compounds as putative CatC inhibitors. One compound, IcatCXPZ-01 ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4'-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide)) was identified as a potent inhibitor of both human and rodent CatC. In mice, pharmacokinetic studies revealed that IcatCXPZ-01 accumulated in the bone marrow reaching levels suitable for CatC inhibition. Subcutaneous administration of IcatCXPZ-01 in a monoclonal anti-collagen antibody induced mouse model of rheumatoid arthritis resulted in statistically significant anti-arthritic activity with persistent decrease in arthritis scores and paw thickness.
Assuntos
Antiasmáticos/química , Antiasmáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Catepsina C/antagonistas & inibidores , Catepsina C/metabolismo , Animais , Antiasmáticos/farmacologia , Cristalografia por Raios X/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Relação Estrutura-Atividade , Células U937RESUMO
Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.
Assuntos
Trifosfato de Adenosina/metabolismo , Flavobacteriaceae/enzimologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Conformação Proteica , Multimerização ProteicaRESUMO
Chondroadherin (CHAD) is a cartilage matrix protein that mediates the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine-rich repeats (LRR) flanked by cysteine-rich domains. CHAD makes important interactions with collagen as well as with cell-surface heparin sulfate proteoglycans and α2ß1 integrins. The integrin-binding site is located in a region of hitherto unknown structure at the C-terminal end of CHAD. Peptides based on the C-terminal human CHAD (hCHAD) sequence have shown therapeutic potential for treating osteoporosis. This article describes a still-unconventional structure solution by phasing with de novo models, the first of a ß-rich protein. Structure determination of hCHAD using traditional, though nonsystematic, molecular replacement was unsuccessful in the hands of the authors, possibly owing to a combination of low sequence identity to other LRR proteins, four copies in the asymmetric unit and weak translational pseudosymmetry. However, it was possible to solve the structure by generating a large number of de novo models for the central LRR domain using Rosetta and multiple parallel molecular-replacement attempts using AMPLE. The hCHAD structure reveals an ordered C-terminal domain belonging to the LRRCT fold, with the integrin-binding motif (WLEAK) being part of a regular α-helix, and suggests ways in which experimental therapeutic peptides can be improved. The crystal structure itself and docking simulations further support that hCHAD dimers form in a similar manner to other matrix LRR proteins.
RESUMO
Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.
Assuntos
Trifosfato de Adenosina/metabolismo , Pseudomonas aeruginosa/enzimologia , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Ribonucleotídeo Redutases/genéticaRESUMO
Protein conformational switches have many useful applications but are difficult to design rationally. Here we demonstrate how the isoenergetic energy landscape of higher-order coiled coils can enable the formation of an oligomerization switch by insertion of a single destabilizing element into an otherwise stable computationally designed scaffold. We describe a de novo designed peptide that was discovered to switch between a parallel symmetric pentamer at pH 8 and a trimer of antiparallel dimers at pH 6. The transition between pentamer and hexamer is caused by changes in the protonation states of glutamatic acid residues with highly upshifted pKa values in both oligomer forms. The drastic conformational change coupled with the narrow pH range makes the peptide sequence an attractive candidate for introduction of pH sensing into other proteins. The results highlight the remarkable ability of simple-α helices to self-assemble into a vast range of structural states.
Assuntos
Peptídeos/química , Proteínas/química , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376-83. ©2016 AACR.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a ß-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.
Assuntos
Dobramento de Proteína , Desdobramento de Proteína , Proteínas/química , Termodinâmica , Algoritmos , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , TemperaturaRESUMO
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.
Assuntos
Cisteína/química , Ribonucleotídeo Redutases/química , Thermotoga maritima/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)ß(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.
Assuntos
Sítio Alostérico/fisiologia , DNA/biossíntese , Desoxirribonucleotídeos/metabolismo , Taxa de Mutação , Ribonucleotídeo Redutases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Domínio Catalítico/fisiologia , Nucleotídeos de Desoxiadenina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Lactobacillus leichmannii/genética , Lactobacillus leichmannii/metabolismo , Modelos Moleculares , Conformação Proteica , Ribonucleotídeo Redutases/química , Leveduras/genética , Leveduras/metabolismoRESUMO
Class II ribonucleotide reductases (RNR) catalyze the formation of an essential thiyl radical by homolytic cleavage of the Co-C bond in their adenosylcobalamin (AdoCbl) cofactor. Several mechanisms for the dramatic acceleration of Co-C bond cleavage in AdoCbl-dependent enzymes have been advanced, but no consensus yet exists. We present the structure of the class II RNR from Thermotoga maritima in three complexes: (i) with allosteric effector dTTP, substrate GDP, and AdoCbl; (ii) with dTTP and AdoCbl; (iii) with dTTP, GDP, and adenosine. Comparison of these structures gives the deepest structural insights so far into the mechanism of radical generation and transfer for AdoCbl-dependent RNR. AdoCbl binds to the active site pocket, shielding the substrate, transient 5'-deoxyadenosyl radical and nascent thiyl radical from solution. The e-propionamide side chain of AdoCbl forms hydrogen bonds directly to the α-phosphate group of the substrate. This interaction appears to cause a "locking-in" of the cofactor, and it is the first observation of a direct cofactor-substrate interaction in an AdoCbl-dependent enzyme. The structures support an ordered sequential reaction mechanism with release or relaxation of AdoCbl on each catalytic cycle. A conformational change of the AdoCbl adenosyl ribose is required to allow hydrogen transfer to the catalytic thiol group. Previously proposed mechanisms for radical transfer in B12-dependent enzymes cannot fully explain the transfer in class II RNR, suggesting that it may form a separate class that differs from the well-characterized eliminases and mutases.
Assuntos
Cobamidas/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Thermotoga maritima/enzimologia , Regulação Alostérica , Cristalografia por Raios X , Guanosina Difosfato/metabolismo , Modelos Moleculares , Thermotoga maritima/química , Nucleotídeos de Timina/metabolismoRESUMO
Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well as in vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined, but increases in both Vmax and S0.5 seem to be involved, as recently shown for human HSL. In this study, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than unphosphorylated HSL. Taken together, our results show that HSL increases its hydrophobic nature upon phosphorylation by PKA. This suggests that PKA phosphorylation induces a conformational change that increases the exposed hydrophobic surface and thereby facilitates binding of HSL to the lipid substrate.