RESUMO
The small GTPase Ras plays an important role in intracellular signal transduction and functions as a molecular switch. In this study, we used a photoresponsive protein as the molecular regulatory device to photoregulate Ras GTPase activity. Photo zipper (PZ), a variant of the photoresponsive protein Aureochrome1 developed by Hisatomi et al. was incorporated into the C-terminus of Ras as a fusion protein. The three constructs of the Ras-PZ fusion protein had spacers of different lengths between Ras and PZ. They were designed using an Escherichia coli expression system. The Ras-PZ fusion proteins exhibited photoisomerization upon blue light irradiation and in the dark. Ras-PZ dimerized upon light irradiation. Moreover, Ras GTPase activity, which is accelerated by the Ras regulators guanine nucleotide exchange factors and GTPase-activating proteins, is controlled by photoisomerization. It has been suggested that light-responsive proteins are applicable to the photoswitching of the enzymatic activity of small GTPases as photoregulatory molecular devices.
Assuntos
Proteínas ras , Proteínas ras/metabolismo , Proteínas ras/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Luz , Escherichia coli/metabolismo , Escherichia coli/genética , Processos FotoquímicosRESUMO
The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, thereby functioning as a molecular switch. Ras forms a nanocluster on the plasma membrane by modifying lipids in the hypervariable region (HVR) at the C-terminus to exhibit physiological functions. In this study, we demonstrated that chemical modification of cysteine residues in HVR with caged compounds (instead of lipidation) induces multimerization of H-Ras. The sulfhydryl-reactive caged compound, 2-nitrobenzyl bromide, was stoichiometrically incorporated into the cysteine residue of HVR and induced the formation of the Ras multimer. Light irradiation induced the elimination of the 2-nitrobenzyl group, resulting in the conversion of the multimer to a monomer. Size-exclusion chromatography coupled with high-performance liquid chromatography and small-angle x-ray scattering analysis revealed that H-Ras forms a pentamer. Electron microscopic observation of the multimer showed a circular ring shape, which is consistent with the structure estimated from x-ray scattering. The shape of the multimer may reflect the physiological state of Ras. It was suggested that the multimerization and monomerization of H-Ras were controlled by modification with a caged compound in HVR under light irradiation.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5'-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5-KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111-Glu116 and Ile135-Asp149, α3: Asn206-Thr226; L5: Gly117-Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.
Assuntos
Biflavonoides , Inibidores Enzimáticos , Garcinia , Cinesinas , Plantas Medicinais , Fuso Acromático , Animais , Camundongos , Adenosina Trifosfatases/antagonistas & inibidores , Biflavonoides/química , Biflavonoides/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Garcinia/química , Cinesinas/antagonistas & inibidores , Cinesinas/química , Cinesinas/metabolismo , Mitose/efeitos dos fármacos , Simulação de Acoplamento Molecular/métodos , Plantas Medicinais/química , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismoRESUMO
The mitotic kinesin Eg5 is a plus-end directed homotetrameric molecular motor essential for the formation of bipolar spindles during cell division. Kinesin Eg5 is overexpressed in cancer cells and hence considered as a target for cancer therapy; the inhibitors specific for Eg5 have been developed as anticancer drugs. In this study, we synthesized a novel functional photoresponsive inhibitor composed of spiropyran and azobenzene derivatives to control Eg5 function with multistage inhibitory activity accompanied by the formation of different isomerization states. The photochromic inhibitor spiropyran-sulfo-azobenzene (SPSAB) exhibited three isomerization states: spiro (SP)-trans, merocyanine (MC)-cis and MC-trans, upon exposure to visible light, ultraviolet and in the dark, respectively. SPSAB-induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with photoisomerization among the three states. Among the three isomerization states of SPSAB, the SP-trans isomer showed potent inhibitory activity at an IC50 value of 30 µM in the basal ATPase assay. MC-trans and MC-cis exhibited less inhibitory activity at IC50 values of 38 and 86 µM, respectively. The results demonstrated that the novel photochromic inhibitor enabled precise control of Eg5 function at three different levels using light irradiation.
Assuntos
Compostos Azo/farmacologia , Benzopiranos/farmacologia , Indóis/farmacologia , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Mitose , Nitrocompostos/farmacologia , Adenosina Trifosfatases/metabolismo , Compostos Azo/química , Compostos Azo/metabolismo , Benzopiranos/química , Benzopiranos/metabolismo , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Indóis/química , Indóis/metabolismo , Isomerismo , Cinesinas/química , Luz , Substâncias Luminescentes/farmacologia , Microtúbulos/metabolismo , Nitrocompostos/química , Nitrocompostos/metabolismo , Ligação ProteicaRESUMO
An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle.
RESUMO
Among 40 plant-derived biflavonoids with inhibitory potential against Eg5, morelloflavone from Garcinia dulcis leaves was selected for further testing based on in silico analysis of binding modes, molecular interactions, binding energies and functional groups that interact with Eg5. Computational models predicted that morelloflavone binds the putative allosteric pocket of Eg5, within the cavity surrounded by amino acid residues of Ile-136, Glu-116, Glu-118, Trp-127, Gly-117, Ala-133, Glu-215, Leu-214 and Tyr-211. Binding energy was -8.4 kcal/mol, with a single hydrogen bond formed between morelloflavone and Tyr-211. The binding configuration was comparable to that of a reference inhibitor, S-trityl-L-cysteine. Subsequent biochemical analysis in vitro confirmed that morelloflavone inhibited both the basal and microtubule-activated ATPase activity of Eg5 in a manner that does not compete with ATP binding. Morelloflavone also suppressed Eg5 gliding along microtubules. These results suggest that morelloflavone binds the allosteric binding site in Eg5 and thereby inhibits ATPase activity and motor function of Eg5.
Assuntos
Biflavonoides/farmacologia , Inibidores Enzimáticos/farmacologia , Cinesinas/antagonistas & inibidores , Mitose , Biflavonoides/química , Inibidores Enzimáticos/química , Garcinia/química , Humanos , Cinesinas/metabolismo , Modelos Moleculares , Estrutura Molecular , Folhas de Planta/químicaRESUMO
Mitotic kinesin Eg5 plays an important physiological role in cell division. Several small-molecule inhibitors of Eg5 are the focus of cancer therapies. Azobenzene is a photochromic compound exhibiting cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. Photochromic compounds of azobenzene derivatives, mimicking Eg5-specific inhibitors of STLC, indicated photoreversible inhibitory effects on Eg5 ATPase activity; however, the photoreversible switching efficiency was not significant. This study presents a novel synthesized photochromic Eg5 inhibitor 2, 3-bis[(2,5-dioxo-1-{4-[(E)-2-phenyldiazen-1-yl]phenyl}pyrrolidin-3-yl)sulfanyl] butanedioic acid (BDPSB), which is composed of two azobenzenes. BDPSB exhibited cis-trans isomerization with UV and VIS light irradiation. The trans form of BDPSB significantly inhibited microtubule-dependent ATPase activity of Eg5, with an IC50 of 74 µM. Cis BDPSB showed weak effects on the microtubule-dependent ATPase activity. The results suggest that the novel photochromic Eg5 inhibitor BDPSB, which exhibits highly efficient photoswitching, shows a switch 'ON' and 'OFF' behaviour with VIS and UV light irradiation.
Assuntos
Adenosina Trifosfatases/metabolismo , Compostos Azo/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Cinesinas/química , Luz , Pirrolidinas/química , Succinatos/química , Proteínas de Xenopus/metabolismo , Compostos Azo/química , Cinesinas/metabolismo , Estrutura Molecular , Fotoquímica , Pirrolidinas/farmacologia , Coloração e Rotulagem , Succinatos/farmacologiaRESUMO
In this study, we synthesized a novel photochromic inhibitor of the mitotic kinesin Eg5, which is composed of the photochromic compound spiropyran to photo-control the function of Eg5. The compound (S)-2, 3-dispiropyran propionic acid (DSPPA) exhibits reversible spiropyran-merocyanine photo-isomerization upon irradiation with visible or ultra-violet light. DSPPA induced reversible changes in the inhibitory effect on Eg5 ATPase and motor activities, which correlates with the spiropyran-merocyanine photo-isomerization. Microtubule-dependent ATPase activity was significantly more inhibited by the spiropyran isomer of DSPPA than by the merocyanine isomer. Additionally, an in vitro motility assay revealed that the microtubule gliding velocity was reduced more by the spiropyran isomer than by the merocyanine isomer. This indicates that the spiropyran derivative may be useful in regulating the function of the mitotic kinesin.
Assuntos
Benzopiranos/química , Indóis/química , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Luz , Mitose/efeitos dos fármacos , Nitrocompostos/química , Raios Ultravioleta , Adenosina Trifosfatases/metabolismo , Animais , Isomerismo , Camundongos , Microtúbulos/enzimologiaRESUMO
A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Corantes Fluorescentes/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/síntese química , Guanosina Trifosfato/química , Cinética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/metabolismoRESUMO
Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.
Assuntos
Membrana Celular/química , Gramicidina/química , Líquidos Iônicos/química , Membrana Celular/metabolismo , Gramicidina/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Íons/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos , Simulação de Dinâmica MolecularRESUMO
The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing conformational changes in nucleotide-binding motifs have been well studied at the molecular level. Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans forms upon exposure to ultraviolet and visible light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the nucleotide-binding motif of Ras to regulate the GTPase activity by photoirradiation. We prepared four Ras mutants (G12C, Y32C, I36C, and Y64C) that have a single reactive cysteine residue in the nucleotide-binding motif. PAM was stoichiometrically incorporated into the cysteine residue of the mutants. The PAM-modified mutants exhibited reversible alterations in GTPase activity, nucleotide exchange rate, and interaction between guanine nucleotide exchange factor and Ras, accompanied by photoisomerization upon exposure to ultraviolet and visible light irradiation. The results suggest that incorporation of photochromic molecules into its nucleotide-binding motif enables photoreversible control of the function of the small G protein Ras.
RESUMO
Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.
Assuntos
Cisteína/análogos & derivados , Cinesinas/antagonistas & inibidores , Luz , Mitose/efeitos dos fármacos , Processos Fotoquímicos , Absorção , Animais , Compostos Azo/química , Cisteína/química , Cisteína/farmacologia , Isomerismo , Cinesinas/metabolismo , Cinética , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Análise Espectral , Sus scrofaRESUMO
In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4-1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4-1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40-50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Nucleotídeos/metabolismo , Marcadores de Spin , Adenosina Trifosfatases/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Microtúbulos/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Estrutura Secundária de Proteína , RotaçãoRESUMO
Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essential for bipolar spindle formation during eukaryotic cell division. Loop L5 of mitotic kinesin Eg5 is a key region determining ATPase activity and motor function. Photochromic molecules undergo reversible isomerization in response to ultraviolet and visible light irradiation. We introduced three kinds of photochromic molecules, 4-phenylazomaleinanil (PAM), 4-(N-(2-iodoacetyl)amino)-4'-(N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (IATAB) and 3,3-dimethyl-1-(2-(2-iodoacetoxy)ethyl)-3H-1,2-dihydroindole-2-spiro-2'-(2H)-6'-nitrochromene (IASP) into L5 to control the Eg5 ATPase activity using light irradiation. We prepared five kinesin Eg5 motor domain mutants, E116C, E118C, Y125C, W127C and D130C, which contained a single reactive cysteine residue in loop L5. The ability of S-trityl-l-cysteine (STLC), a specific Eg5 inhibitor, to inhibit E116C, W127C and D130C was significantly reduced. The photochromic molecules were stoichiometrically incorporated into the cysteine residues in L5 of mutants. W127C and D130C modified with IASP exhibited reversible ATPase activity alterations when subjected to light irradiation-induced photoisomerization. The two IASP modified mutants also demonstrated photocontrolled alterations following treatment with STLC. Additionally, the ATPase activity of the mutant D130C modified with PAM could be photocontrolled. Our findings demonstrate that incorporation of photochromic molecules into the key region of loop L5 facilitates the photocontrol of the function of kinesin Eg5.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Mitose , Sondas Moleculares/metabolismo , Processos Fotoquímicos , Compostos de Sulfidrila/metabolismo , Absorção , Animais , Compostos Azo/química , Compostos Azo/metabolismo , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Cinética , Camundongos , Microtúbulos/metabolismo , Sondas Moleculares/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Análise Espectral , Sus scrofaRESUMO
The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have "on-off" control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca(2+)-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca(2+)-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.
Assuntos
Cálcio/química , Calmodulina/química , Cinesinas/química , Engenharia de Proteínas/métodos , Animais , Calmodulina/genética , Cinesinas/genética , Camundongos , Movimento (Física) , Mutação , Multimerização ProteicaRESUMO
We previously demonstrated that the rice kinesin K16, which belongs to the kinesin-7 subfamily, has unique enzymatic properties and atomic structure within key functional regions. In this study, we focused on a novel rice plant kinesin, K23, which also belongs to the kinesin-7 subfamily. The biochemical characterization of the K23 motor domain (K23MD) was studied and compared with the rice kinesin K16 and other related kinesins. K23 exhibits â¼45-fold (1.3 Pi mol(-1) site mol(-1) s(-1)) lower microtubule-dependent ATPase activity than conventional kinesins, whereas its affinity for microtubules is comparable with conventional kinesins. MgADP-free K23 is unstable compared with the unusually stable MgADP-free K16MD. The enzymatic properties of K23MD are somewhat different from those of K16. We used a fluorescent ATP analogue 2'(3')-O-(N'-methylanthraniloyl)-ATP (mant-ATP) for the kinetic characterization of K23. The fluorescence of mant-ATP was not significantly altered during its hydrolysis by K23. However, significant fluorescence resonance energy transfer (FRET) between mant-ATP and W21 in the motor domain was observed. The kinetic study using FRET revealed that K23 has unique kinetic characteristics when compared with other kinesins.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Cinesinas/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Triptofano/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinesinas/química , Cinesinas/classificação , Cinesinas/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Triptofano/metabolismo , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismoRESUMO
A novel non-nucleotide fluorescent ATP analogue, N-methylanthraniloylamideethyl triphosphate (MANTTP), was designed and synthesized for kinetic studies with ATPases. The interaction of MANTTP with myosin ATPase was characterized. MANTTP was used as a substrate of myosin ATPase, and acceleration of actin-dependent hydrolysis was observed. The fluorescence property of MANTTP was not greatly affected by its binding to the ATPase site of myosin. In contrast, during MANTTP hydrolysis, significant fluorescence resonance energy transfer (FRET) was observed between MANTTP and intrinsic tryptophan residues in the myosin motor domain. Binding of MANTTP and formation of a ternary complex with a myosin-N-methylanthraniloylamideethyl diphosphate (MANTDP)-Pi analogue, which may mimic ATPase transient states, were monitored by FRET. The kinetic parameters of MANTTP binding to myosin and MANTDP release from the ATPase site were determined using a stopped-flow apparatus and compared with those of other ATP analogues. This novel fluorescent ATP analogue was shown to be applicable for kinetic analysis of ATPases.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Fluorescência , Miosinas/química , Miosinas/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Galinhas , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Modelos Moleculares , Estrutura Molecular , Músculo Esquelético/enzimologiaRESUMO
Genomic analysis predicted that the rice (Oryza sativa var. japonica) genome encodes at least 41 kinesin-like proteins including the novel kinesin O12, which is classified as a kinesin-14 family member. O12 has a calponin homology (CH) domain that is known as an actin-binding domain. In this study, we expressed the functional domains of O12 in Escherichia coli and determined its enzymatic characteristics compared with other kinesins. The microtubule-dependent ATPase activity of recombinant O12 containing the motor and CH domains was significantly reduced in the presence of actin. Interestingly, microtubule-dependent ATPase activity of the motor domain was also affected by actin in the absence of the CH domain. Our findings suggest that the motor activity of the rice plant-specific kinesin O12 may be regulated by actin.
Assuntos
Cinesinas/química , Oryza , Proteínas de Plantas/química , Proteínas Recombinantes/química , Actinas/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Ligação ao Cálcio/genética , Cinesinas/classificação , Cinesinas/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Filogenia , Proteínas de Plantas/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , CalponinasRESUMO
Biochemical studies revealed that the novel rice plant-specific kinesin K16 has several unique enzymatic characteristics as compared to conventional kinesins. The ADP-free form of K16 is very stable, whereas the ADP-free form of conventional kinesins is labile. In the present study, the crystal structure of the novel rice kinesin motor domain (K16MD) complexed with Mg-ADP was determined at 2.4 Å resolutions. The overall structure of K16MD is similar to that of conventional kinesin motor domains, as expected from the high amino acid sequence similarity (43.2%). However, several unique structures in K16 were observed. The position and length of the L5, L11, and L12 loops, which are key functional regions, were different from those observed in conventional kinesins. Moreover, the neck-linker region of the ADP-bound K16MD showed an ordered conformation at a position quite different from that previously observed in conventional kinesins. These structural differences may reflect the unique enzymatic characteristics of rice kinesin K16.