RESUMO
Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 µM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Miosinas/metabolismo , Conformação Proteica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-AtividadeRESUMO
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
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
Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. The kinesin family is classified into subfamilies. Kinesin species derived from vertebrates have been well characterized. In contrast, plant kinesins have yet to be adequately characterized. In this study, we expressed the motor domain of a novel rice plant-specific kinesin, K16, in Escherichia coli, and then determined its enzymatic characteristics and compared them with those of kinesin 1. Our findings demonstrated that the rice kinesin motor domain has different enzymatic properties from those of well known kinesin 1.