RESUMEN
In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500â MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.
Asunto(s)
Ciclamas , Zinc , Zinc/química , Zinc/metabolismo , Cristalografía por Rayos X , Ciclamas/química , Ciclamas/farmacología , Regulación Alostérica , Presión , Unión Proteica , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Humanos , Sitios de UniónRESUMEN
The 31P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the 31P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH2p and GTPγS was measured in the absence and presence of Mg2+-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B 1 was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg2+·GMP and Mg2+·GppNHp the second order pressure coefficients are positive. To describe the data of Mg2+·GppCH2p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg2+ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure 31P NMR data on free Mg2+-GDP and Mg2+-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.
Asunto(s)
Nucleótidos de Guanina/química , Espectroscopía de Resonancia Magnética , Concentración de Iones de Hidrógeno , Marcaje Isotópico , Espectroscopía de Resonancia Magnética/métodos , MetalesRESUMEN
Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation, and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins (1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined. Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies. Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin filaments.
Asunto(s)
Schistosoma mansoni/química , Septinas/química , Animales , Sitios de Unión , Calorimetría , Catálisis , Membrana Celular/metabolismo , Cristalografía por Rayos X , Escherichia coli/metabolismo , GTP Fosfohidrolasas/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Hidrólisis , Magnesio/química , Espectroscopía de Resonancia Magnética , Nucleótidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Termodinámica , Agua/químicaRESUMEN
Ciliopathies are human diseases arising from defects in primary or motile cilia. The small G-protein Arl13B (ADP-ribosylation factor-like 13B) localizes to microtubule doublets of the ciliary axoneme and is mutated in Joubert syndrome. Its GDP/GTP mechanistic cycle and the effect of its mutations in patients with Joubert syndrome remain elusive. In the present study we applied high resolution structural and biochemical approaches to study Arl13B. The crystal structure of Chlamydomonas rheinhardtii Arl13B, comprising the G-domain and part of its unique C-terminus, revealed an incomplete active site, and together with biochemical data the present study accounts for the absence of intrinsic GTP hydrolysis by this protein. The structure shows that the residues representing patient mutations R79Q and R200C are involved in stabilizing important intramolecular interactions. Our studies suggest that Arg79 is crucial for the GDP/GTP conformational change by stabilizing the large two-residue register shift typical for Arf (ADP-ribosylation factor) and Arl subfamily proteins. A corresponding mutation in Arl3 induces considerable defects in effector and GAP (GTPase-activating protein) binding, suggesting a loss of Arl13B function in patients with Joubert syndrome.
Asunto(s)
Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Enfermedades Cerebelosas/genética , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Mutación/genética , Retina/anomalías , Anomalías Múltiples , Secuencia de Aminoácidos , Cerebelo/anomalías , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
The small GTPase Ras is an essential component of signal transduction pathways within the cell, controlling proliferation, differentiation, and apoptosis. Only in the GTP-bound form does Ras interact strongly with effector molecules such as Raf-kinase, thus acting as a molecular switch. In the GTP-bound form, Ras exists in a dynamic equilibrium between at least two distinct conformational states, 1(T) and 2(T), offering different functional properties of the protein. Zn2+-cyclen is a typical state 1(T) inhibitor; i.e., it interacts selectively with Ras in conformational state 1(T), a weak effector binding state. Here we report that active K-Ras4B, which is prominently found to be mutated in human tumors, exhibits a dynamic equilibrium like H-Ras, which can be modulated by Zn2+-cyclen. The titration experiments of Ras with Zn2+-cyclen indicate a cooperatively coupled binding of the ligands to the two interaction sites on Ras that could be identified for H-Ras previously. Our data further indicate that as in state 2(T) where induced fit produces the substate 2(T)* after effector binding, a corresponding substate 1(T)* can be detected at the state 1(T) mutant Ras(T35A). The interaction of Zn2+-cyclen with Ras not only shifts the equilibrium toward the weak effector binding state 1(T) but also perturbs the formation of substate 1(T)*, thus enhancing the inhibitory effect. Although Zn2+-cyclen shows an affinity for Ras in only the millimolar range, its potency of inhibition corresponds to a competitive state 2 inhibitor with micromolar binding affinity. Thus, the results demonstrate the mode of action and potency of this class of allosteric Ras inhibitors.
Asunto(s)
Complejos de Coordinación/farmacología , Compuestos Heterocíclicos con 1 Anillo/farmacología , Proteínas ras/antagonistas & inhibidores , Ciclamas , Guanosina Trifosfato/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Quinasas raf/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Allosteric interactions: Metal(II) cyclens inhibit Ras-effector interactions by stabilizing a weak effector-binding state of Ras, stateâ 1(T), and binding directly in the active site. The novel stateâ (1T) inhibitor Zn(2+)-BPA (BPA = bis(2-picolyl)amine) binds outside the nucleotide binding pocket but nevertheless allosterically stabilizes stateâ 1(T) and thus inhibits the Ras-Raf interaction.
Asunto(s)
Aminas/química , Complejos de Coordinación/química , Ácidos Picolínicos/química , Zinc/química , Proteínas ras/antagonistas & inhibidores , Proteínas ras/química , Sitios de Unión , Cobre/química , Diseño de Fármacos , Modelos Moleculares , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.
RESUMEN
Guanine nucleotide binding proteins (GNB-proteins) play an essential role in cellular signaling, acting as molecular switches, cycling between the inactive, GDP-bound form and the active, GTP-bound form. It has been shown that conformational equilibria also exist within the active form of GNB-proteins between conformational states with different functional properties. Here we present (31)P NMR data on ADP ribosylation factor 1 (Arf1), a GNB-protein involved in Golgi traffic, promoting the coating of secretory vesicles. To investigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety of commonly used GTP analogues, namely, GppCH(2)p, GppNHp, and GTPγS, were analyzed. To gain deeper insight into the conformational state of active Arf1, we titrated with Cu(2+)-cyclen and GdmCl and formed the complex with the Sec7 domain of nucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, from (31)P NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This state seems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effector domain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward a conformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchange factors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effector interacting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuning mechanism of regulation and thereby an interesting target for the modulation of protein activity.
Asunto(s)
Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/metabolismo , Guanosina Trifosfato/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Cobre/metabolismo , Ciclamas , Guanidina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Compuestos Heterocíclicos/metabolismo , Humanos , Magnesio , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Isótopos de Fósforo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Treonina/genética , VolumetríaRESUMEN
The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.
Asunto(s)
Guanosina Trifosfato/química , Proteínas Proto-Oncogénicas p21(ras)/química , Sustitución de Aminoácidos , Animales , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Humanos , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , RatasRESUMEN
(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran.
Asunto(s)
Proteínas de Unión al GTP/química , Compuestos Heterocíclicos/química , Dominio Catalítico , Ciclamas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras.Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras.GTP versus Ras.GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.
Asunto(s)
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas raf/metabolismo , Biología Computacional , Humanos , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Mutación/genética , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Quinasas raf/genéticaRESUMEN
Folding and function of proteins are two aspects of proteins which are usually considered as basically unrelated phenomena that are optimized by evolution independently. From the funnel model of folding/unfolding and the associated energy landscape, we infer the paradigm that the minimum number of folding intermediates is determined by the number of all functional states of a protein ("essential" folding intermediates). Here, we demonstrate the supposed fundamental link using the Ras protein complexed with the GTP analogue GppNHp that occurs in two structural states coexisting in solution. State 2 was shown earlier to represent the effector interacting state, and the function of state 1 was hitherto unknown. By (31)P NMR spectroscopy, we demonstrate that state 1 represents the conformation interacting with guanine nucleotide exchange factors (GEFs). Denaturation experiments of the protein with a chaotropic reagent show that both functional states coexist during folding and unfolding. Application of high pressure represents another perturbation of the energy landscape, leading to an increased population of the state 1 as observed by NMR spectroscopy. The specific volume difference between the two states DeltaV(12) is 17.2 +/- 0.5 mL mol(-1), indicating that state 1 represents a more open conformation of the protein. The free energies of stabilization for state 1 and state 2 at 278 K can be determined as 8.3 and 9.8 kJ mol(-1), respectively.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Guanosina Trifosfato/química , Proteínas ras/química , Entropía , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Transducción de Señal , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Guanine nucleotide binding proteins, such as Ras proteins, play a pivotal role in maintaining the regular life cycle of cells. The involvement of Ras mutants in the progress of cancer has attracted many efforts to find detection methods for Ras activity. In this study we present a luminescent microwell plate assay for monitoring GTPase activity of Ras proteins. The luminescence intensity of the Tb-norfloxacin complex is influenced by nucleoside phosphates as well as by inorganic phosphates. Real-time kinetics of the GTPase activity of wild-type Ras and Ras mutants can be monitored online. The effect of a GTPase activating protein as well as of a downstream effector (Ras-binding domain of human Raf-1) on the GTPase activity of different Ras mutants is examined. In contrast to other methods, this assay does not require the use of radioactively labeled substrates or chromatographic separation steps. Moreover, the application of fluorescently labeled GTP substrates which often interfere with enzymatic activity can be avoided. This in vitro assay can serve as a model system for the screening of regulators affecting the GTPase activity of Ras proteins.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Luminiscencia , Compuestos Organometálicos/química , Terbio/química , Proteínas ras/metabolismo , Calibración , GTP Fosfohidrolasas/química , Guanosina/química , Cinética , Mediciones Luminiscentes , Norfloxacino/química , Fosfatos/química , Reproducibilidad de los Resultados , Terbio/análisis , Factores de Tiempo , Proteínas ras/químicaRESUMEN
The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.
Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas ras/química , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Termodinámica , Proteínas ras/metabolismoRESUMEN
Liquid-state 31P NMR spectroscopy is a well-established method for the study of guanine nucleotide-binding proteins (GNB proteins) such as the proto-oncogene Ras. Solid-state 31P NMR spectroscopy could meanwhile also be used to study microcrystalline samples of Ras as well as its partial loss-of-function mutants Ras(T35S) and Ras(T35A). However, solid-state NMR studies of the latter mutants in complex with effector molecules such as RalGDS or Raf kinase were so far prevented, since it has been impossible to crystallize these complexes yet. The aim of the present contribution is to make such complexes accessible to solid-state 31P NMR spectroscopy by the application of precipitation methods. The complex formed by Ras(T35S) and Raf kinase is preserved during precipitation. In contrast, the weakly bound complex of Ras(T35S) with RalGDS is dissociated or at least perturbed by the precipitation procedure. Solid-state 31P NMR experiments on precipitates of these complexes deliver spectra of high resolution and signal-to-noise ratio which allows the application of two-dimensional techniques. Precipitates prepared using polyethylene glycol 6000 (PEG) as precipitant were found to exhibit spectra of maximum resolution and signal-to-noise ratio. Interestingly, the 31P signal due to the alpha-phosphate of GppNHp bound to Ras(T35S) in crystalline samples or aged precipitates has a significantly different isotropic chemical shift than in the liquid state or in freshly prepared precipitates. This directly indicates that the crystal structure differs from the equilibrium solution structure at least in the neighborhood of the alpha-phosphate group.
Asunto(s)
Nucleótidos de Guanina/química , Quinasas raf/química , Factor de Intercambio de Guanina Nucleótido ral/química , Proteínas ras/química , Mutación , Resonancia Magnética Nuclear Biomolecular , Polietilenglicoles , Unión Proteica , Proteínas ras/genéticaRESUMEN
High pressure NMR spectroscopy is a powerful method for identifying rare conformational states of proteins from the pressure response of their chemical shifts. Many proteins have bound adenine nucleotides at their active centers, in most cases in a complex with Mg2+-ions. The 31P NMR signals of phosphate groups of the nucleotides can be used as probes for conformational transitions in the proteins themselves. For distinguishing protein specific pressure effects from trivial pressure responses not due to the protein interaction, data of the pressure response of the free nucleotides must be available. Therefore, the pressure response of 31P chemical shifts of the adenine nucleotides AMP, ADP, and ATP and their Mg2+-complexes has been determined at pH values several units distant from the respective pK-values. It is clearly non-linear for most of the resonances. A negative first order pressure coefficient B1 was determined for all 31P resonances except Mg2+·AMP indicating an upfield shift of the resonances with pressure. The smallest and largest negative values are obtained for the α-phosphate group of ADP and ß-phosphate group of Mg2+·ATP with -0.32 and -4.59ppm/GPa, respectively. With exception of the α-phosphate group of Mg2+·AMP the second order pressure coefficients are positive leading to a saturation like behaviour. The pressure response of the adenine nucleotides is similar but not identical to that observed earlier for guanine nucleotides. The obtained data show that the chemical shift pressure response of the different phosphate groups is rather different dependent on the position of phosphate group in the nucleotide and the nucleotide used.
Asunto(s)
Nucleótidos de Adenina/química , Adenosina Difosfato/química , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Magnesio/química , Espectroscopía de Resonancia Magnética , Fósforo/química , PresiónRESUMEN
Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.
Asunto(s)
Guanosina Trifosfato/análogos & derivados , Proteínas ras/química , Proteínas ras/genética , Sitios de Unión , Cristalización , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fósforo/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soluciones , Proteínas ras/metabolismoAsunto(s)
Complejos de Coordinación/química , Compuestos Heterocíclicos/química , Proteínas ras/química , Sitios de Unión , Cristalografía por Rayos X , Ciclamas , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , ZincRESUMEN
Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.