Takayuki Ariga

Principal role of the arginine finger in rotary catalysis of F1-ATPase

Background: The role of arginine finger of F1-ATPase in acceleration of hydrolysis and cooperativity is controversial. Results: An arginine finger mutant (αR364K) of thermophilic Bacillus PS3 F1-ATPase showed slow, successive, unidirectional rotations with tight chemomechanical coupling. Conclusion: The arginine finger of F1-ATPase accelerates ATP hydrolysis and is dispensable for cooperativity. Significance: The arginine finger is involved in the rotary catalysis of F1-ATPase. F1-ATPase (F1) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α3β3 stator ring. The 3 catalytic sites of F1 reside on the interface of the α and β subunits of the α3β3 ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F1 remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F1 with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F1 showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F1 carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.

Simultaneous measurement of nucleotide occupancy and mechanical displacement in myosin-V, a processive molecular motor.

Adenosine triphosphate (ATP) turnover drives various processive molecular motors and adenosine diphosphate (ADP) release is a principal transition in this cycle. Biochemical and single molecule mechanical studies have led to a model in which a slow ADP release step contributes to the processivity of myosin-V. To test the relationship between force generation and ADP release, we utilized optical trapping nanometry and single molecule total internal reflection fluorescence imaging for simultaneous and direct observation of both processes in myosin-V. We found that ADP was released 69 +/- 5.3 ms after force generation and displacement of actin, providing direct evidence for slow ADP release. As proposed by several previous studies, this slow ADP release probably ensures processivity by prolonging the strong actomyosin state in the ATP turnover cycle.

Measurement system for simultaneous observation of myosin V chemical and mechanical events

Myosin V is an actin-based processive molecular motor driven by the chemical energy of ATP hydrolysis. Although the chemo-mechanical coupling in processive movement has been postulated by separate structural, mechanical and biochemical studies, no experiment has been able to directly test these conclusions. Therefore the relationship between ATP-turnover and force generation remains unclear. Currently, the most direct method to measure the chemo-mechanical coupling in processive motors is to simultaneously observe ATP-turnover cycles and displacement at the single molecule level. In this study, we developed a simultaneous measurement system suitable for mechanical and chemical assays of myosin V in order to directly elucidate its chemo-mechanical coupling.

The concerted nature between three catalytic subunits driving the F1 rotary motor.

F(1), a rotational molecular motor, shows strong cooperativity during ATP catalysis when driving the rotation of the central gamma subunit surrounded by the alpha(3)beta(3) subunits. To understand how the three catalytic beta subunits cooperate to drive rotation, we made a hybrid F(1) containing one or two mutant beta subunits with altered catalytic kinetics and observed its rotations. Analysis of the asymmetric stepwise rotations elucidated a concerted nature inside the F(1) complex where all three beta subunits participate to rotate the gamma subunit with a 120 degrees phase. In addition, observing hybrid F(1) rotations at various solution conditions, such as ADP, P(i) and the ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) provides additional information for each elementary event. This novel experimental system, which combines single molecule observations and biochemical methods, enables us to dynamically visualize the catalytic coordination inside active enzymes and shed light on how biological machines provide unidirectional functions and rectify information from stochastic reactions.

Single molecule FRET observation of kinesin-1's head-tail interaction on microtubule

Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.