Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Autonomous assembly and disassembly of gliding molecular robots regulated by a DNA-based molecular controller.

In recent years, there has been a growing interest in engineering dynamic and autonomous systems with robotic functionalities using biomolecules. Specifically, the ability of molecular motors to convert chemical energy to mechanical forces and the programmability of DNA are regarded as promising components for these systems. However, current systems rely on the manual addition of external stimuli, limiting the potential for autonomous molecular systems. Here, we show that DNA-based cascade reactions can act as a molecular controller that drives the autonomous assembly and disassembly of DNA-functionalized microtubules propelled by kinesins. The DNA controller is designed to produce two different DNA strands that program the interaction between the microtubules. The gliding microtubules integrated with the controller autonomously assemble to bundle-like structures and disassemble into discrete filaments without external stimuli, which is observable by fluorescence microscopy. We believe this approach to be a starting point toward more autonomous behavior of motor protein-based multicomponent systems with robotic functionalities.

Overexpression of the flagellar motor protein MotB sensitizes Bacillus subtilis to aminoglycosides in a motility-independent manner.

The flagellar motor proteins, MotA and MotB, form a complex that rotates the flagella by utilizing the proton motive force (PMF) at the bacterial cell membrane. Although PMF affects the susceptibility to aminoglycosides, the effect of flagellar motor proteins on the susceptibility to aminoglycosides has not been investigated. Here, we found that MotB overexpression increased susceptibility to aminoglycosides, such as kanamycin and gentamicin, in Bacillus subtilis without affecting swimming motility. MotB overexpression did not affect susceptibility to ribosome-targeting antibiotics other than aminoglycosides, cell wall-targeting antibiotics, DNA synthesis-inhibiting antibiotics, or antibiotics inhibiting RNA synthesis. Meanwhile, MotB overexpression increased the susceptibility to aminoglycosides even in the motA-deletion mutant, which lacks swimming motility. Overexpression of the MotB mutant protein carrying an amino acid substitution at the proton-binding site (D24A) resulted in the loss of the enhanced aminoglycoside-sensitive phenotype. These results suggested that MotB overexpression sensitizes B. subtilis to aminoglycosides in a motility-independent manner. Notably, the aminoglycoside-sensitive phenotype induced by MotB requires the proton-binding site but not the MotA/MotB complex formation.

Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence.

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

The pathogenic roles of the p.R130S prestin variant in DFNB61 hearing loss.

DFNB61 is a recessively inherited nonsyndromic hearing loss caused by mutations in SLC26A5, the gene that encodes the voltage-driven motor protein, prestin. Prestin is abundantly expressed in the auditory outer hair cells that mediate cochlear amplification. Two DFNB61-associated SLC26A5 variants, p.W70X and p.R130S, were identified in patients who are compound heterozygous for these nonsense and missense changes (SLC26A5W70X/R130S ). Our recent study showed that mice homozygous for p.R130S (Slc26a5R130S/R130S ) suffer from hearing loss that is ascribed to significantly reduced motor kinetics of prestin. Given that W70X-prestin is nonfunctional, compound heterozygous Slc26a5R130S/- mice were used as a model for human SLC26A5W70X/R130S . By examining the pathophysiological consequences of p.R130S prestin when it is the sole allele for prestin protein production, we determined that this missense change results in progressive outer hair cell loss in addition to its effects on prestin's motor action. Thus, this study defines the pathogenic roles of p.R130S prestin and identifies a limited time window for potential clinical intervention. KEY POINTS: The voltage-driven motor protein, prestin, is encoded by SLC26A5 and expressed abundantly in cochlear outer hair cells (OHCs). The importance of prestin for normal hearing was demonstrated in mice lacking prestin; however, none of the specific SLC26A5 variants identified to date in human patients has been experimentally demonstrated to be pathogenic. In this study we used both cell lines and a mouse model to define the pathogenic role of compound heterozygous p.W70X (c.209G>A) and p.R130S (c.390A>C) SLC26A5 variants identified in patients with moderate to profound hearing loss. As in patients, mice carrying one copy of p.R130S Slc26a5 showed OHC dysfunction and progressive degeneration, which results in congenital progressive hearing loss. This is the first functional study reporting pathogenic SLC26A5 variants and pointing to the presence of a therapeutic time window for potential clinical interventions targeting the affected OHCs before they are lost.

Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor.

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

"Turbo-Charged" DNA Motors with Optimized Sequence Enable Single-Molecule Nucleic Acid Sensing.

DNA motors that consume chemical energy to generate processive mechanical motion mimic natural motor proteins and have garnered interest due to their potential applications in dynamic nanotechnology, biosensing, and drug delivery. Such motors translocate by a catalytic cycle of binding, cleavage, and rebinding between DNA "legs" on the motor body and RNA "footholds" on a track. Herein, we address the well-documented trade-off between motor speed and processivity and investigate how these parameters are controlled by the affinity between DNA legs and their complementary footholds. Specifically, we explore the role of DNA leg length and GC content in tuning motor performance by dictating the rate of leg-foothold dissociation. Our investigations reveal that motors with 0 % GC content exhibit increased instantaneous velocities of up to 150 nm/sec, three-fold greater than previously reported DNA motors and comparable to the speeds of biological motor proteins. We also demonstrate that the faster speed and weaker forces generated by 0 % GC motors can be leveraged for enhanced capabilities in sensing. We observe single-molecule sensitivity when programming the motors to stall in response to the binding of nucleic acid targets. These findings offer insights for the design of high-performance DNA motors with promising real-world biosensing applications.

Effect of small molecular crowders on dynamics of kinesin molecular motors.

Kinesin is a motor protein that can convert chemical energy of ATP hydrolysis into mechanical energy of moving processively on microtubules. Apart from the load and ATP concentration affecting the dynamics of the motor such as velocity, run length, dissociation rate, etc., the increase of solution viscosity by supplementing crowding agents of low molecular weight into the buffer can also affect the dynamics. Here, based on our proposed model for the chemomechanical coupling of the kinesin motor, a systematically theoretical study of the motor dynamics under the variation of the viscosity and load is presented. Both the load on the motor's stalk and that on one of the two heads are considered. The theoretical results provide a consistent explanation of the available contradictory experimental results, with some showing that increasing viscosity decreases sensitively the velocity whereas others showing that increasing viscosity has little effect on the velocity. The theoretical results reproduce quantitatively the puzzling experimental data showing that under different directions of the load on the stalk, increasing viscosity has very different effects on the change of run length or dissociation rate. The theoretical results predict that in both the pure and crowded buffers the dependence of the run length on the load acting one of the two heads has very different feature from that on the load acting on the stalk.

Ion-Powered Rotary Motors: Where Did They Come from and Where They Are Going?

Molecular motors are found in many living organisms. One such molecular machine, the ion-powered rotary motor (IRM), requires the movement of ions across a membrane against a concentration gradient to drive rotational movement. The bacterial flagellar motor (BFM) is an example of an IRM which relies on ion movement through the stator proteins to generate the rotation of the flagella. There are many ions which can be used by the BFM stators to power motility and different ions can be used by a single bacterium expressing multiple stator variants. The use of ancestral sequence reconstruction (ASR) and functional analysis of reconstructed stators shows promise for understanding how these proteins evolved and when the divergence in ion use may have occurred. In this review, we discuss extant BFM stators and the ions that power them as well as recent examples of the use of ASR to study ion-channel selectivity and how this might be applied to further study of the BFM stator complex.

Ion selectivity and rotor coupling of the Vibrio flagellar sodium-driven stator unit.

Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the flagellar rotor remained largely unclear. Here, we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA prime PomA for clockwise rotation. We propose that a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our study provides mechanistic insights for understanding ion selectivity and rotor incorporation of the stator unit of the bacterial flagellum.

Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

Waiting Time Distributions in Hybrid Models of Motor-Bead Assays: A Concept and Tool for Inference.

In single-molecule experiments, the dynamics of molecular motors are often observed indirectly by measuring the trajectory of an attached bead in a motor-bead assay. In this work, we propose a method to extract the step size and stalling force for a molecular motor without relying on external control parameters. We discuss this method for a generic hybrid model that describes bead and motor via continuous and discrete degrees of freedom, respectively. Our deductions are solely based on the observation of waiting times and transition statistics of the observable bead trajectory. Thus, the method is non-invasive, operationally accessible in experiments and can, in principle, be applied to any model describing the dynamics of molecular motors. We briefly discuss the relation of our results to recent advances in stochastic thermodynamics on inference from observable transitions. Our results are confirmed by extensive numerical simulations for parameters values of an experimentally realized F1-ATPase assay.

The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips.

Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.

Prestin's fast motor kinetics is essential for mammalian cochlear amplification.

Prestin (SLC26A5)-mediated voltage-driven elongations and contractions of sensory outer hair cells within the organ of Corti are essential for mammalian cochlear amplification. However, whether this electromotile activity directly contributes on a cycle-by-cycle basis is currently controversial. By restoring motor kinetics in a mouse model expressing a slowed prestin missense variant, this study provides experimental evidence acknowledging the importance of fast motor action to mammalian cochlear amplification. Our results also demonstrate that the point mutation in prestin disrupting anion transport in other proteins of the SLC26 family does not alter cochlear function, suggesting that the potential weak anion transport of prestin is not essential in the mammalian cochlea.

Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo.

The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

Measurements of the Ion Channel Activity of the Transmembrane Stator Complex in the Bacterial Flagellar Motor.

The bacterial flagellum is driven by a rotational motor located at the base of the flagellum. The stator unit complex conducts cations such as protons (H+) and sodium ions (Na+) along the electrochemical potential across the cytoplasmic membrane and interacts with the rotor to generate the rotational force. Escherichia coli and Salmonella have the H+-type stator complex, which serves as a transmembrane H+ channel that couples H+ flow through an ion channel to torque generation whereas Vibrio and some Bacillus species have the Na+-type stator complex. In this chapter, we describe how to measure the ion conductivity of the transmembrane stator complex over-expressed in E. coli cells using fluorescent indicators. Intensity measurements of fluorescent indicators using either a fluorescence spectrophotometer or microscope allow quantitative detection of changes in the intracellular ion concentrations due to the ion channel activity of the transmembrane protein complex.

Purification of the Na+-Driven PomAB Stator Complex and Its Analysis Using ATR-FTIR Spectroscopy.

The flagellar motor of marine Vibrio is driven by the sodium-motive force across the inner membrane. The stator complex, consisting of two membrane proteins PomA and PomB, is responsible for energy conversion in the motor. To understand the coupling of the Na+ flux with torque generation, it is essential to clearly identify the Na+-binding sites and the Na+ flux pathway through the stator channel. Although residues essential for Na+ flux have been identified by using mutational analysis, it has been difficult to observe Na+ binding to the PomAB stator complex. Here we describe a method to monitor the binding of Na+ to purified PomAB stator complex using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. This method demonstrates that Na+-binding sites are formed by critical aspartic acid and threonine residues located in the transmembrane segments of PomAB.

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

Rotary biomolecular motor-powered supramolecular colloidal motor.

Cells orchestrate the motion and force of hundreds of protein motors to perform various mechanical tasks over multiple length scales. However, engineering active biomimetic materials from protein motors that consume energy to propel continuous motion of micrometer-sized assembling systems remains challenging. Here, we report rotary biomolecular motor-powered supramolecular (RBMS) colloidal motors that are hierarchically assembled from a purified chromatophore membrane containing FOF1-ATP synthase molecular motors, and an assembled polyelectrolyte microcapsule. The micro-sized RBMS motor with asymmetric distribution of FOF1-ATPases can autonomously move under light illumination and is collectively powered by hundreds of rotary biomolecular motors. The propulsive mechanism is that a transmembrane proton gradient generated by a photochemical reaction drives FOF1-ATPases to rotate for ATP biosynthesis, which creates a local chemical field for self-diffusiophoretic force. Such an active supramolecular architecture endowed with motility and biosynthesis offers a promising platform for intelligent colloidal motors resembling the propulsive units in swimming bacteria.

Renal injury associated with MYH9 disorder with 5773delG mutation: A case report.

A 35-year-old man with persistent urine abnormalities and renal dysfunction was referred to our hospital. May-Hegglin anomaly was suspected, and a renal biopsy showed focal segmental glomerulosclerosis (FSGS) with IgA deposition. Electron microscopy revealed foot process effacements and intense bleb-like morphological changes in podocytes. Nonmuscle myosin heavy chain IIA (NMMHCIIA) staining of granulocytes revealed a localized, type II pattern, and genomic DNA sequencing of MYH9 exon 40 revealed MYH9 5773delG mutation (c.5773delG [p.(Asp1925Thrfs*23)]). Podocytes were significantly stained by an antibody specific for NMMHC-IIA abnormalities associated with this mutation. Colocalization observation of vimentin and NMMHC-IIA demonstrated a diminished form of NMMHC-IIA in podocytes. Taking these observations into account, it was determined that the present case was likely associated with MYH9 disorder. Treatment was started with olmesartan, followed by methylprednisolone pulse therapy 3 times bi-monthly. Finally, the patient began hemodialysis 18 months later. This is the first known report of renal phenotype expression associated with this MYH9 mutation. FSGS can occur in association with MYH9 mutations at the 3' regions, such as exon 40. Abnormal expression or metabolism of NMMHC-IIA in podocytes might be related to the formation of FSGS lesions due to this MYH9 mutation.