Probing protein-protein interactions with drag flow: a case study of F-actin and tropomyosin.

Tropomyosins are central regulators of the actin cytoskeleton, controlling the binding and activity of the other actin binding proteins. The interaction between tropomyosin and actin is quite unique: single tropomyosin dimers bind weakly to actin filaments but get stabilised by end-to-end attachment with neighbouring tropomyosin dimers, forming clusters which wrap around the filament. Force spectroscopy is a powerful approach for studying protein-protein interactions, but classical methods which usually pull with pN forces on a single protein pair, are not well adapted to tropomyosins.

Development of clinically viable non-muscle myosin II small molecule inhibitors.

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by a lack of selective tools. The most prototypical non-selective inhibitor inactivates both NMII and cardiac muscle myosin II (CMII), a key regulator of heart function.

Modulation of Filopodial Myosin Function.

MyTH-FERM (MF) myosins are essential for filopodia formation in both Metazoa and Amoebozoa ( DdMyo7; mammalian Myo10) but their roles in filopodia formation are not fully understood. Taking advantage of a mutation in the highly conserved actin binding interface of another MF myosin, the () mutation of Myo15A that reduces its function, the impact of altering the myosin-F-actin interaction on filopodia formation was investigated. The mutation, a D to G substitution (Gong et al, 2022, Sci Adv), was introduced into DdMyo7 or Myo10 and filopodia formation assessed by quantitative analysis of number, length and myosin tip enrichment.

Aficamten is a small-molecule cardiac myosin inhibitor designed to treat hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of the sarcomere resulting in excessive cardiac contractility. The first-in-class cardiac myosin inhibitor, mavacamten, improves symptoms in obstructive HCM. Here we present aficamten, a selective small-molecule inhibitor of cardiac myosin that diminishes ATPase activity by strongly slowing phosphate release, stabilizing a weak actin-binding state.

Reassessing the unifying hypothesis for hypercontractility caused by myosin mutations in hypertrophic cardiomyopathy.

Human β-cardiac myosin exists in an ON-state where both myosin heads are accessible for interaction with actin, and an OFF-state where the heads are folded back onto their own coiled-coil tail, interacting with each other via an interacting-heads motif (IHM). Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin cause hypercontractility of the heart. Nine years ago, a unifying hypothesis proposed that hypercontractility caused by myosin HCM-associated mutations is primarily due to an increase in the number of ON-state myosin molecules, rather than altered fundamental alterations of functional myosin parameters such as intrinsic motor force, its velocity of movement along actin, or its ATPase turnover rate, all of which impact power output.

Omecamtiv mecarbil and Mavacamten target the same myosin pocket despite opposite effects in heart contraction.

Inherited cardiomyopathies are common cardiac diseases worldwide, leading in the late stage to heart failure and death. The most promising treatments against these diseases are small molecules directly modulating the force produced by β-cardiac myosin, the molecular motor driving heart contraction. Omecamtiv mecarbil and Mavacamten are two such molecules that completed phase 3 clinical trials, and the inhibitor Mavacamten is now approved by the FDA.

A weak coupling mechanism for the early steps of the recovery stroke of myosin VI: A free energy simulation and string method analysis.

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever-arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever-arm and ATP hydrolysis are coupled.

Uncovering the Relationship Between Genes and Phenotypes Beyond the Gut in Microvillus Inclusion Disease.

Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID.

Omecamtiv mecarbil and Mavacamten target the same myosin pocket despite antagonistic effects in heart contraction.

Inherited cardiomyopathies are amongst the most common cardiac diseases worldwide, leading in the late-stage to heart failure and death. The most promising treatments against these diseases are small-molecules directly modulating the force produced by β-cardiac myosin, the molecular motor driving heart contraction. Two of these molecules that produce antagonistic effects on cardiac contractility have completed clinical phase 3 trials: the activator and the inhibitor .

How myosin VI traps its off-state, is activated and dimerizes.

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6.

Nucleotide-free structures of KIF20A illuminate atypical mechanochemistry in this kinesin-6.

KIF20A is a critical kinesin for cell division and a promising anti-cancer drug target. The mechanisms underlying its cellular roles remain elusive. Interestingly, unusual coupling between the nucleotide- and microtubule-binding sites of this kinesin-6 has been reported, but little is known about how its divergent sequence leads to atypical motility properties.

Mechanism of small molecule inhibition of Plasmodium falciparum myosin A informs antimalarial drug design.

Malaria results in more than 500,000 deaths per year and the causative Plasmodium parasites continue to develop resistance to all known agents, including different antimalarial combinations. The class XIV myosin motor PfMyoA is part of a core macromolecular complex called the glideosome, essential for Plasmodium parasite mobility and therefore an attractive drug target. Here, we characterize the interaction of a small molecule (KNX-002) with PfMyoA.

Cryo-EM structure of the folded-back state of human β-cardiac myosin.

To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human β-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.

Cryo-EM structure of the folded-back state of human β-cardiac myosin.

During normal levels of exertion, many cardiac muscle myosin heads are sequestered in an off-state even during systolic contraction to save energy and for precise regulation. They can be converted to an on-state when exertion is increased. Hypercontractility caused by hypertrophic cardiomyopathy (HCM) myosin mutations is often the result of shifting the equilibrium toward more heads in the on-state.

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism.

The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding are only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp.

Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures.

The human mutant cardiac α-actins p.A295S or p.R312H and p.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth.

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in , the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo.

VASP-mediated actin dynamics activate and recruit a filopodia myosin.

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation.

The many roles of myosins in filopodia, microvilli and stereocilia.

Filopodia, microvilli and stereocilia represent an important group of plasma membrane protrusions. These specialized projections are supported by parallel bundles of actin filaments and have critical roles in sensing the external environment, increasing cell surface area, and acting as mechanosensors. While actin-associated proteins are essential for actin-filament elongation and bundling in these protrusions, myosin motors have a surprising role in the formation and extension of filopodia and stereocilia and in the organization of microvilli.

Biological nanomotors, driving forces of life.

Life is driven by awe-inspiring coordinated movements observed in cells and tissues. In each cell, nm-size molecular motor proteins contribute to these movements as they power numerous mechanical processes with precision and complex orchestration. For the multiple functions that an eukaryotic cell accomplish, motility is essential both at molecular and cellular scales.

The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity.

Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now.