A shift of dynamic equilibrium between the KIT active and inactive states causes drug resistance

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 μs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.     

A slow interconversion between active and inactive states of the (Na-K)ATPase.

We have examined slow changes in the rate of ATP hydrolysis for purified dog kidney Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] at various concentrations of free Mg2+, Mg-ATP, K+, and Na+. The effect of these ligands on the rate of ATP hydrolysis is explained by a rapid binding step determining the initial rate of turnover followed by a slow conformational change. Inactivation of enzyme stored in the presence of ethylenediaminetetraacetic acid occurs upon adding free Mg2+, Mg-ATP, and K+; reactivation may be achieved if the concentration of these ligands is reduced. Because of the slow conformational change, the affinities for ligands affecting inactivation are time dependent. A model is presented to explain the effects of free Mg2+ and Ma-ATP on (Na-K)ATPase activity.     

Protein structural changes associated with active and inactive states of hydrogenase from Thiocapsa roseopersicina

Electrophoretic and isoelectrofocusing behavior of the hydrogenase from Thiocapsa roseopersicina under various conditions revealed remarkable properties of this enzyme: there are two active forms which differ in their molecular masses as well as in oxygen sensitivity; the apparent molecular masses of the active hydrogenase forms (90 and 49 kDa) differ considerably from those in the inactive state (64, 34, and 15 kDa); the active forms and some of the inactivated ones can be transformed into each other reversibly; urea can unfold the 64 and 34 kDa proteins but not the 49 kDa form at room temperature; the pI of these proteins are different in the presence of urea. The results suggest large rearrangements in the hydrogenase protein structure which are associated with the enzymatically active and inactive states. It is concluded that reversible formation of disulfide bonds cannot be the major cause for maintaining the enzyme conformation. Strong hydrophobic interactions are suggested to be primarily responsible for the structural stability and for the rearrangements.     

Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects--enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization of the retinal ligand--despite the extensive differences in sequences between mGluRs and rhodopsin.     

Structural characterization of the active and inactive states of Src kinase in solution by small-angle X-ray scattering

Src kinase plays an important role in several signaling and regulation mechanisms in vivo. Enzymatic activity is tightly regulated through the phosphorylation and dephosphorylation of tyrosine 527, which is placed at the C-terminal tail. Here, we have addressed domain rearrangements involved in the regulation mechanism of Src kinase in solution using small-angle X-ray scattering. In the phosphorylated wild-type form of Src kinase corresponding to the inactive state of the protein, a single conformation compatible with a closed crystallographic structure was found in solution. In the Y527F point mutant representing the active state, analysis of scattering data reveals an equilibrium between two differently populated conformations differing in the radius of gyration by 5 Å. The major species (85% of the total population) presents a closed conformation indistinguishable from the crystallographic structure of the inactive state. The minor species (15% of the total population) is an open conformation similar to the crystallographic structure in the active state. The latter structure has the SH3, SH2, and SH2-catalytic domain linker assembled as a pseudo-two-domain protein. The regulation model emerging from this study, including at least three different conformational states, allows the tight regulation of the enzyme without compromising fast response in the presence of natural targets.     

Structural features of the inactive and active states of the melanin-concentrating hormone receptors: insights from molecular simulations

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of "helix 8" to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.     

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.     

Golden hamster myoid cells during active and inactive states of spermatogenesis: correlation of testosterone levels with structure

Myoid cells were examined quantitatively in adult golden hamsters with active spermatogenesis and compared with hamsters in which the testes were regressed due to a modification in the light-dark cycle. A detailed morphometric study was undertaken utilizing animals previously examined. The cell-surface area and volumes of most organelles were not significantly different in animals which were gonadally active as compared with regressed animals. A slight, but significant, increase in nuclear volume (31%) and a slight, but significant, decrease (28%) in cell volume were recorded for regressed animals. The total volume of pinocytotic vesicles was increased dramatically (approximately threefold) in active animals in comparison with inactive animals (P less than 0.01), indicating that an increase in non-specific transport across the myoid cell is associated with spermatogenic activity. Intravascularly injected horseradish peroxidase was capable of entering pinocytotic vesicles in both active and inactive animals. Plasma luteinizing hormone (LH) as well as plasma and testicular testosterone levels were weakly (r = 0.64, 0.68, and 0.65, respectively), but significantly (P less than 0.05), correlated with cell size. Plasma and testicular testosterone were correlated with the total volume of pinocytotic vesicles (r = 0.74 and 0.68, respectively). The data indicate that although the rat myoid cell possesses receptors for testosterone, there are few structural manifestations of the hamster myoid cell that correlate well with testosterone levels. Thus, the hamster myoid cell differs from two other hormone-responsive somatic cells in the testis, the Sertoli cell and the Leydig cell, that show dramatic structural alterations with changes in gonadal activity and striking correlations of structural features with functional measures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histidine-49 is necessary for the pH-dependent transition between active and inactive states of the bovine F1-ATPase inhibitor protein

The role of the histidyl residue at position 49 (H49) of the bovine mitochondrial F₁-ATPase inhibitor protein (F₁I) was examined by site-directed mutagenesis. Six amino acids (Q, E, K, V, L, and I) were substituted for H49 and the activities of the resulting inhibitor proteins were characterized with respect to pH. Each of the six mutations abolished the pH sensitivity which is characteristic of wild-type F₁I. At pH 8.0, each of the mutations caused an increase in apparent maximum inhibition and a decrease in apparent K_i relative to wild type. At pH 6.7 the hydrophilic substitutions had little effect on apparent K_i, while the hydrophobic substitutions caused increases of 3.5- to 8.5-fold relative to wild type. The ratios of apparent K_i at pH 8.0 to apparent K_i at pH 6.7 were in the range of 0.5 to 1.6 for the mutants, whereas the wild-type value is 15.0. The mutations appear to shift the equilibrium between active and inactive conformations of F₁I toward the active state. We find that H49 is required by F₁I for sensitivity to pH and that it may facilitate the transition between active and inactive states of F₁I. A possible role for H49 in the stabilization of the inactive state through participation in a multivalent complex with Zn²⁺ is also discussed.     

Gangliosides of active and inactive neuroblastoma clones.

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones). The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.     

Large-scale production of a disulfide-stabilized constitutively active mutant opsin

Previous studies of constitutively activated mutants of opsin in the absence of chromophore were carried out in crude cell membranes because such mutants could not be recovered in a detergent-solubilized form in the active state. We employed a strategy in which a stabilizing disulfide bond allowed for successful purification of a constitutively activated mutant opsin, N2C/E113Q/M257Y/D282C, solubilized in nonionic detergent from mammalian cell culture. The purified mutant opsin is able to activate transducin to a higher degree than opsin and may prove useful for future structural studies of the active state of GPCRs.     

Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Fixation of transporters in the active or inactive state

A strategy is presented for fixing regulated transport systems in the functional or non-functional state. The advantages of this approach include the ability to distinguish between sensor and effector systems, the capacity to study transporters independently of their activating mechanisms, and the possibility of identifying membrane polypeptides that participate in the regulatory process.     

Signaling states of rhodopsin: absorption of light in active metarhodopsin II generates an all-trans-retinal bound inactive state

Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A(max) = 500 nm (P(500)), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P(500) does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P(500) is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P(500) and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a "second switch" between active and inactive conformations that operates by photolysis but without isomerization around the C(11)-C(12) double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal.