Shinsaku Maruta

Characterization of a novel rice kinesin O12 with a calponin homology domain

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.

Photocontrol of Calmodulin Interaction with Target Peptides using Azobenzene Derivative

Calmodulin (CaM), a physiologically important Ca(2+)-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short alpha-helix. Each of the globular domains has two Ca(2+)-binding sites, the EF hands. CaM undergoes a conformational change upon binding to Ca(2+), which enables it to bind to specific proteins for specific responses. Here, we successfully photocontrolled CaM binding to its target peptide using the photochromic compound N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. In order to specifically incorporate PAM, CaM mutants having reactive cysteine residues in the functional region were prepared; PAM was stoichiometrically incorporated into the cysteine residues in these mutants. Further, we prepared the target peptide, M13, fused with yellow fluorescent protein (YFP) to monitor the CaM-M13 peptide interaction. The binding of the PAM-CaM mutants, N60C, D64C and M124C, to M13-YFP was reversibly photocontrolled upon UV-VIS light irradiation at appropriate Ca(2+) concentrations.

Biochemical characterization of the novel rice kinesin K23 and its kinetic study using fluorescence resonance energy transfer between an intrinsic tryptophan residue and a fluorescent ATP analogue

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 ∼45-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.

Crystallographic analysis reveals a unique conformation of the ADP-bound novel rice kinesin K16.

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.

Synthesis of a novel fluorescent non-nucleotide ATP analogue and its interaction with myosin ATPase

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.

Kinesin–Calmodulin fusion protein as a molecular shuttle

In this study, we developed a molecular shuttle with reversible cargo-loading system by using calmodulin (CaM) and M13 peptide. We designed a kinesin (K560) chimera protein with CaM fused at the C-terminal tail region of K560 (K560-CaM). K560-CaM was expressed using an Escherichia coli expression system and purified. Its ATPase activity and microtubule gliding velocity were almost in a similar range as those of the wild-type kinesin. Ca(2+)-dependent reversible binding of K560-CaM and M13 peptide was monitored by size-exclusion-HPLC. Rotary shadowing and electron microscopy revealed tetrameric configuration of K560-CaM in the absence of Ca(2+), while both dimeric and tetrameric configurations in the presence of Ca(2+). Further, Ca(2+)-dependent change in the configuration of K560-CaM was monitored by size-exclusion-HPLC and analytical ultracentrifugation. Finally, by total internal reflection fluorescence microscopy, we successfully observed that K560-CaM transported quantum dot-conjugated M13 peptide along the microtubule in the presence of Ca(2+).

Characterization of the Plant-Specific Rice Kinesins

Kinesin is a motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics and signal transduction. Kinesin converts chemical energy from ATP into mechanical force. Kinesin family is classified into some subfamilies. Some species of kinesin derived from vertebrate have been well studied. However, not so many studies for kinesins of plants have been done yet. Recently, the genome sequences of rice were completed. Bioinformatical analyses revealed that at least 41 kinesin-related proteins were encoded on the rice genome. In this study, we focused on the two rice kinesins; 1. O12 that has a calponin homology domain, 2. K23 that belongs to At1 subfamily in kinesin-7. The cDNAs of the kinesin motor domain was subcloned into expression vector pET and transformed into E. coli BL21 (DE3). kinesin motor domains were expressed and purified by Co-NTA column. The biochemical characterizations of the two rice kinesins were studied. The microtubule-dependent ATPase activity of the two rice kinesins motor domains were 30∼60-fold lower than that of conventional kinesin. Kinetic analyses using stopped-flow demonstrated that ATP binding to O12 in the absence of microtubule was extremely slow compared with that of conventional kinesin. While, ATP binding to K23 was not accelerated by microtubule. Furthermore, interestingly ATPase activity of O12 in the absence microtubule regulated by actin. The O12-tail fused with GFP was observed to localize in the actin filament in the onion cell. The two plant specific rice kinesin O12 and K23 were shown to have unique enzymatic properties.

Synthesis of Photochromic ATP Analogue and its Interaction with Motor Proteins

Azobenzene is one of the photochromic molecules, which undergoes rapid and reversible transitions between the cis isomer and trans isomer by visible and ultra-violet light irradiation. We have been trying to control the activities of motor proteins using the photochromic molecules as photo-regulatory devices. We have recently demonstrated that microtubules dependent ATPase activity of the kinesin modified by azobenzene derivative was regulated by UV-VIS light irradiation. However, it was not so easy to incorporate the photochromic molecules into the functional site of motor proteins without altering the native enzymatic properties.In the present study, we have designed the novel ATP analogues consist of photochromic molecules in order to photo-regulate the motor protein kinesin without their chemical modification. It is expected that the ATP analogues induce the reversible conformational change in the active site by alternate UV-VIS light irradiation. We have synthesized non-nucleotide ATP analogue composed of azobenzene derivative, Phenylazobenzoic-aminoethyl -triphosphate (PABATP). PABATP showed UV/VIS light absorption spectral change accompanied by transition between cis and trans in a similar manner observed in azobenzene. PABATP was hydrolyzed by conventional kinesin and the hydrolysis rate was activated by microtubules. It has been demonstrated that the cis isomer and trans isomer perform differently on a microtubule gliding motility assay. We have also examined the formation of kinesin-PABADP-Pi analogues (BeFn, AlF4-, Vi) which may mimic the transient states in ATPase cycle.