Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.