A significant challenge in engineering molecular motors is designing mechanisms to coordinate the motion between multiple domains of the motor so as to bias random motion. For bipedal motors, this challenge takes the form of coordinating the movement of the biped’s legs, so they can move in a synchronized fashion. To address this problem, we have constructed an autonomous DNA bipedal nanorobot that coordinates the action of its two legs by coupling room temperature thermal energy with the catalysis of metastable DNA fuel strand hybrids. This coupling leads to a chemically ratcheted unidirectional walk along a DNA track. By crosslinking aliquots of the walker covalently to its track in successive walking states, we demonstrate that the walker can complete a full walking cycle on a track whose length could be extended for longer walks.

Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minus-end directed myosin VI motor domain in rigor (4.6 Å) and Mg-ADP (5.5 Å) states bound to F-actin. Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.

Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3–5 and modified natural protein motors6–10 have been developed in separate complementary strategies for achieving tunable and controllable motor function. Integrating protein and nucleic acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number, and composition of tethered protein motors11–15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport, and can be dynamically controlled using programmed transitions in lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy, and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10–20 nm s−1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

Phase separation of RNA-binding proteins via multivalent interactions between aromatic/polar-rich disordered domains contributes to the formation of functional cytoplasmic granules and nuclear puncta. These domains have also been identified as the nucleators of neuronal inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We use atomic resolution nuclear magnetic resonance spectroscopy approaches for visualizing low complexity domain structure and interactions along the pathway from monomer, to liquid-liquid phase separated state, to static aggregates and hydrogels. We show that the low complexity domain of RNA-binding protein Fused in Sarcoma (FUS LC) remains disordered even within liquid phase separated states and recruits unphosphorylated RNA-polymerase II C-terminal domain into the liquid phase separated state, adding a potential explanation for FUS LC transcriptional activation in cancer. Importantly, phase separation is reversible and is modulated by ionic strength and interaction with RNA, distinguishing these assemblies from static inclusions. In contrast, we show that liquid-liquid phase separation of TDP-43 is mediated in part by a-helical assembly and extension. Some ALS-associated mutations disrupt helix-helix interaction inhibiting liquidliquid phase separation while leading to enhanced aggregation.

Allosteric mechanisms are fundamental to the operation of biomolecular motors. Recreating the molecular phenomena associated with allostery, from first principles, would help advance the design and construction of synthetic molecular motors, which remain quite simple compared natural motors. In this study, I present a model for generating allosteric interactions using mechanical linkages, which are devices in which flexible nodes are connected by rigid rods. I describe how allosteric information can be communicated between multivalent binding sites on an enzyme when linkages bind or dissociate in a stepwise fashion, which takes place stochastically according to assigned binding rates and partitioned binding energies. This design allows geometric competitions to autonomously push a linkage enzyme through a desired sequence of states, driven by consumption of a fuel. I use the model to emulate the chemical and conformational cycle of a myosin monomer, which is demonstrated by simulating chemical reaction networks of the linkage structures, and by describing how two linkage monomers can be connected together to construct a motor that walks on a track. This work shows how the complex behavior of biomolecular motors can be recapitulated with simple geometric and chemical principles that encode allosteric mechanisms. Because the concepts are material-agnostic, they can potentially be used to design and construct allosteric machines using various chemistries.

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Despite extensive scrutiny of the myosin superfamily, the lack of high-resolution structures of actin-bound states has prevented a complete description of its mechanochemical cycle and limited insight into how sequence and structural diversification of the motor domain gives rise to specialized functional properties. Here we present cryo-EM structures of the unique minusend directed myosin VI motor domain in rigor (4.6 Å ) and Mg-ADP (5.5 Å ) states bound to F-actin.Comparison to the myosin IIC-F-actin rigor complex reveals an almost complete lack of conservation of residues at the actin-myosin interface despite preservation of the primary sequence regions composing it, suggesting an evolutionary path for motor specialization. Additionally, analysis of the transition from ADP to rigor provides a structural rationale for force sensitivity in this step of the mechanochemical cycle. Finally, we observe reciprocal rearrangements in actin and myosin accompanying the transition between these states, supporting a role for actin structural plasticity during force generation by myosin VI.

No abstract