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1.
Sci Rep ; 9(1): 12987, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506493

RESUMO

During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

2.
Nat Commun ; 8(1): 764, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970495

RESUMO

CD22 maintains a baseline level of B-cell inhibition to keep humoral immunity in check. As a B-cell-restricted antigen, CD22 is targeted in therapies against dysregulated B cells that cause autoimmune diseases and blood cancers. Here we report the crystal structure of human CD22 at 2.1 Å resolution, which reveals that specificity for α2-6 sialic acid ligands is dictated by a pre-formed ß-hairpin as a unique mode of recognition across sialic acid-binding immunoglobulin-type lectins. The CD22 ectodomain adopts an extended conformation that facilitates concomitant CD22 nanocluster formation on B cells and binding to trans ligands to avert autoimmunity in mammals. We structurally delineate the CD22 site targeted by the therapeutic antibody epratuzumab at 3.1 Å resolution and determine a critical role for CD22 N-linked glycosylation in antibody engagement. Our studies provide molecular insights into mechanisms governing B-cell inhibition and valuable clues for the design of immune modulators in B-cell dysfunction.The B-cell-specific co-receptor CD22 is a therapeutic target for depleting dysregulated B cells. Here the authors structurally characterize the ectodomain of CD22 and present its crystal structure with the bound therapeutic antibody epratuzumab, which gives insights into the mechanism of inhibition of B-cell activation.


Assuntos
Autoimunidade/imunologia , Linfócitos B/imunologia , Imunidade Humoral/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Anticorpos Monoclonais Humanizados/ultraestrutura , Cristalografia por Raios X , Humanos , Lectinas/imunologia , Microscopia Eletrônica , Terapia de Alvo Molecular , Conformação Proteica , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/ultraestrutura
3.
Nature ; 539(7627): 118-122, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27776355

RESUMO

Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V1 region drives proton translocation through the membrane-embedded VO region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase. We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae, allowing us to obtain a ~3.9-Šresolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac8c'c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.


Assuntos
Microscopia Crioeletrônica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Arginina/química , Arginina/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Hidrólise , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Rotação , Saccharomyces cerevisiae/química
4.
Sci Adv ; 2(7): e1600725, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27532044

RESUMO

Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases.


Assuntos
Microscopia Crioeletrônica , ATPases Vacuolares Próton-Translocadoras/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Thermus thermophilus/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
J Biol Chem ; 291(30): 15641-52, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27226556

RESUMO

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.


Assuntos
Adenocarcinoma , Neoplasias Pulmonares , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Síndrome de Noonan , Neoplasias Urológicas , Proteínas ras , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Substituição de Aminoácidos , Linhagem Celular , Guanosina Trifosfato/química , Humanos , Hidrólise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Domínios Proteicos , Neoplasias Urológicas/genética , Neoplasias Urológicas/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismo
6.
Proc Natl Acad Sci U S A ; 112(21): 6625-30, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25941399

RESUMO

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.


Assuntos
Genes ras , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Humanos , Bicamadas Lipídicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Eletricidade Estática , Fator ral de Troca do Nucleotídeo Guanina/química , Fator ral de Troca do Nucleotídeo Guanina/genética , Fator ral de Troca do Nucleotídeo Guanina/metabolismo
7.
J Biol Chem ; 289(18): 12195-201, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24648513

RESUMO

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g. Ras Q61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g. Rap) or not catalytic (e.g. Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introduction of a bulkier valine side chain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mammalian target of rapamycin (mTOR) signaling.


Assuntos
Glicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Neuropeptídeos/genética , Serina-Treonina Quinases TOR/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Motivos de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Células Cultivadas , Cristalografia por Raios X , Glicina/química , Glicina/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo
9.
J Am Chem Soc ; 135(9): 3367-70, 2013 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-23409921

RESUMO

Like most Ras superfamily proteins, the GTPase domain of Ras homologue enriched in brain (Rheb) is tethered to cellular membranes through a prenylated cysteine in a flexible C-terminal region; however, little is known about how Rheb or other GTPases interact with the membrane or how this environment may affect their GTPase functions. We used NMR methods to characterize Rheb tethered to nanodiscs, monodisperse protein-encapsulated lipid bilayers with a diameter of 10 nm. Membrane conjugation markedly reduced the rate of intrinsic nucleotide exchange, while GTP hydrolysis was unchanged. NMR measurements revealed that the GTPase domain interacts transiently with the surface of the bilayer in two distinct preferred orientations, which are determined by the bound nucleotide. We propose models of membrane-dependent signal regulation by Rheb that shed light on previously unexplained in vivo properties of this GTPase. The study presented provides a general approach for direct experimental investigation of membrane-dependent properties of other Ras-superfamily GTPases.


Assuntos
Bicamadas Lipídicas/química , Proteínas Monoméricas de Ligação ao GTP/química , Nanoestruturas/química , Neuropeptídeos/química , Ressonância Magnética Nuclear Biomolecular , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Estrutura Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo
10.
PLoS One ; 7(11): e48707, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185272

RESUMO

The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Espectroscopia de Ressonância Magnética , Regulação Alostérica , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-Atividade , Temperatura Ambiente
11.
Methods ; 57(4): 473-85, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22750304

RESUMO

The Ras superfamily of small GTPases is a large family of switch-like proteins that control diverse cellular functions, and their deregulation is associated with multiple disease processes. When bound to GTP they adopt a conformation that interacts with effector proteins, whereas the GDP-bound state is generally biologically inactive. GTPase activating proteins (GAPs) promote hydrolysis of GTP, thus impeding the biological activity of GTPases, whereas guanine nucleotide exchange factors (GEFs) promote exchange of GDP for GTP and activate GTPase proteins. A number of methods have been developed to assay GTPase nucleotide hydrolysis and exchange, as well as the activity of GAPs and GEFs. The kinetics of these reactions are often studied with purified proteins and fluorescent nucleotide analogs, which have been shown to non-specifically impact hydrolysis and exchange. Most GAPs and GEFs are large multidomain proteins subject to complex regulation that is challenging to reconstitute in vitro. In cells, the activities of full-length GAPs or GEFs are typically assayed indirectly on the basis of nucleotide loading of the cognate GTPase, or by exploiting their interaction with effector proteins. Here, we describe a recently developed real-time NMR method to assay kinetics of nucleotide exchange and hydrolysis reactions by direct monitoring of nucleotide-dependent structural changes in an isotopically labeled GTPase. The unambiguous readout of this method makes it possible to precisely measure GAP and GEF activities from extracts of mammalian cells, enabling studies of their catalytic and regulatory mechanisms. We present examples of NMR-based assays of full-length GAPs and GEFs overexpressed in mammalian cells.


Assuntos
Extratos Celulares/química , GTP Fosfo-Hidrolases/química , Proteínas Ativadoras de GTPase/química , Fatores de Troca do Nucleotídeo Guanina/química , Animais , Dineínas/química , Ensaios Enzimáticos , Corantes Fluorescentes/química , Guanosina Trifosfato/química , Células HEK293 , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética , Proteínas Monoméricas de Ligação ao GTP/química , Neuropeptídeos/química , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Recombinantes/química , Transdução de Sinais , Transfecção , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/química
12.
Structure ; 20(9): 1528-39, 2012 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-22819219

RESUMO

Rheb, an activator of mammalian target of rapamycin (mTOR), displays low intrinsic GTPase activity favoring the biologically activated, GTP-bound state. We identified a Rheb mutation (Y35A) that increases its intrinsic nucleotide hydrolysis activity ∼10-fold, and solved structures of both its active and inactive forms, revealing an unexpected mechanism of GTP hydrolysis involving Asp65 in switch II and Thr38 in switch I. In the wild-type protein this noncanonical mechanism is markedly inhibited by Tyr35, which constrains the active site conformation, restricting the access of the catalytic Asp65 to the nucleotide-binding pocket. Rheb Y35A mimics the enthalpic and entropic changes associated with GTP hydrolysis elicited by the GTPase-activating protein (GAP) TSC2, and is insensitive to further TSC2 stimulation. Overexpression of Rheb Y35A impaired the regulation of mTORC1 signaling by growth factor availability. We demonstrate that the opposing functions of Tyr35 in the intrinsic and GAP-stimulated GTP catalysis are critical for optimal mTORC1 regulation.


Assuntos
Guanosina Trifosfato/química , Proteínas Monoméricas de Ligação ao GTP/química , Neuropeptídeos/química , Proteínas/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Ligações de Hidrogênio , Hidrólise , Cinética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Termodinâmica
13.
Biophys J ; 102(3): 630-9, 2012 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-22325287

RESUMO

EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (ß2-ß3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , AMP Cíclico/metabolismo , Entropia , Fatores de Troca do Nucleotídeo Guanina/genética , Simulação de Dinâmica Molecular , Mutação , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
J Biol Chem ; 285(8): 5132-6, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20018863

RESUMO

The Ras family of small GTPases control diverse signaling pathways through a conserved "switch" mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2'(3')-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is approximately 10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay.


Assuntos
Guanosina Trifosfato/análogos & derivados , Proteínas Monoméricas de Ligação ao GTP/química , Ressonância Magnética Nuclear Biomolecular/métodos , ortoaminobenzoatos/química , Animais , Guanosina Trifosfato/química , Humanos , Hidrólise , Cinética , Camundongos , Proteínas Recombinantes/química
15.
J Biol Chem ; 284(35): 23682-96, 2009 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-19403523

RESUMO

One of the mechanisms that minimize the aberrant cross-talk between cAMP- and cGMP-dependent signaling pathways relies on the selectivity of cAMP binding domains (CBDs). For instance, the CBDs of two critical eukaryotic cAMP receptors, i.e. protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), are both selectively activated by cAMP. However, the mechanisms underlying their cAMP versus cGMP selectivity are quite distinct. In PKA this selectivity is controlled mainly at the level of ligand affinity, whereas in EPAC it is mostly determined at the level of allostery. Currently, the molecular basis for these different selectivity mechanisms is not fully understood. We have therefore comparatively analyzed by NMR the cGMP-bound states of the essential CBDs of PKA and EPAC, revealing key differences between them. Specifically, cGMP binds PKA preserving the same syn base orientation as cAMP at the price of local steric clashes, which lead to a reduced affinity for cGMP. Unlike PKA, cGMP is recognized by EPAC in an anti conformation and generates several short and long range perturbations. Although these effects do not alter significantly the structure of the EPAC CBD investigated, remarkable differences in dynamics between the cAMP- and cGMP-bound states are detected for the ionic latch region. These observations suggest that one of the determinants of cGMP antagonism in EPAC is the modulation of the entropic control of inhibitory interactions and illustrate the pivotal role of allostery in determining signaling selectivity as a function of dynamic changes, even in the absence of significant affinity variations.


Assuntos
Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/química , Fatores de Troca do Nucleotídeo Guanina/química , Regulação Alostérica , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cinética , Ligantes , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
16.
Biochemistry ; 48(2): 223-5, 2009 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19113833

RESUMO

The binding of allosteric ligands to protein can induce changes to the holoprotein conformation, stability, and activity that have an impact on unfolding dynamics. Herein we report a label-free strategy for determining the dissociation constant of protein-ligand interactions over a wide dynamic range (>10(4), Kd from nano- to millimolar) using capillary electrophoresis that overcomes the constraints of an ideal two-state protein unfolding model. Multivariate analysis of thermodynamic parameters associated with holoprotein unfolding and ligand binding is demonstrated for the classification of cyclic nucleotide analogues that function as allosteric modulators of regulatory proteins, such as the exchange protein directly activated by cAMP (EPAC).


Assuntos
Bioensaio , Ligantes , Análise Multivariada , Proteínas/metabolismo , Termodinâmica , Regulação Alostérica , Fenômenos Biofísicos , Eletroforese Capilar/métodos , Cinética , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
17.
J Biol Chem ; 283(28): 19691-703, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18411261

RESUMO

Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.


Assuntos
AMP Cíclico/química , Fatores de Troca do Nucleotídeo Guanina/química , Modelos Moleculares , Regulação Alostérica/fisiologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Entropia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/genética , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
18.
J Am Chem Soc ; 129(46): 14482-92, 2007 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-17973384

RESUMO

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.


Assuntos
Apoproteínas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de AMP Cíclico/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Alostérica , Apoproteínas/química , Domínio Catalítico , AMP Cíclico/química , Deutério/química , Células Eucarióticas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Hidrogênio/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Receptores de AMP Cíclico/química , Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/metabolismo
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