Biology subjects

Houdusse, A.

Publications and source records attributed to Houdusse, A..

18 recordsLinked to original sources

bioRxiv2026

Plasmodium falciparum Myosin B is a slow motor optimized for force generation

Malaria is a disease caused by apicomplexan parasites of the genus Plasmodium. These organisms express two atypical class-XIV myosins: Myosin A (MyoA), a core component of the glideosome expressed throughout the entire lifecycle, and Myosin B (MyoB), which is restricted to invasive stages and localizes to the apical region. Here, we determine the crystal structure of Plasmodium falciparum MyoB (PfMyoB) in the Rigor state and identify its essential light chain as the same subunit bound to MyoA. Combining structural analysis, molecular dynamics simulations and in vitro kinetic and motility assays, we show that PfMyoB is a slow motor optimized for force production during invasion. The N-terminal extensions of PfMyoA and PfMyoB exert distinct effects on each motor s mechanochemistry. Altogether, these findings reveal how Plasmodium myosins have evolved specialized functions during the complex parasite lifecycle and provide insight into developing multi-target inhibitors of erythrocytic invasion based on the PfMyoA inhibitor KNX-002.

biophysics↗

bioRxiv2026

Myosin-X sorts actin filaments into parallel bundles and tunes barbed-end dynamics

Whether a molecular motor can spontaneously organize actin filaments into bundles has remained an open question despite decades of in vitro investigation. Here, we show that the dimeric myosin-X, essential for filopodia initiation and extension in cells, is capable of sorting actin filaments into parallel bundles, gathering barbed ends within 1-2 micrometers. We observe that myosin-X processivity is comparable on single filaments and on bundles induced by myosin-X or fascin. Upon reaching barbed ends, myosin-X slows down the addition or removal of actin subunits, in a myosin density-dependent manner. Furthermore, the funneling of myosin-X towards the remaining filaments at the bundle tip increases motor density and triggers dynamic clustering. Together, we propose that the motor activity of myosin-X is sufficient to initiate filopodia, independently of passive crosslinkers such as fascin or fimbrin.

biochemistry↗

bioRxiv2025

Modulation of Filopodial Myosin Function

MyTH-FERM (MF) myosins are essential for filopodia formation in both Metazoa and Amoebozoa (Dictyostelium 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 jordan (jd) mutation of Myo15A that reduces its function, the impact of altering the myosin-F-actin interaction on filopodia formation was investigated. The jd 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. The mutation significantly decreased filopodia initiation and tip intensity of both DdMyo7 and Myo10. This is consistent with impairment of myosin-based reorganization of cortical actin at the cortex, with myosins in the initiation complex remaining at the tip as the filopodium extends. The DdMyo7 jd mutation did not affect filopodia length. In the case of Myo10, while the jd mutation reduced Myo10 velocity of intrafilopodial motility by 40%, resulting in reduced levels of myosin at the filopodia tip, the jd mutant did not consistently affect the length of filopodia in different cell lines. This indicates that the major role of Myo10 may not be to promote filopodia elongation by reducing membrane tension, as has been proposed. Rather, the phenotype of the DdMyo7 and Myo10 jd mutants indicates that the major role of evolutionarily diverse filopodial myosins is to re-organize actin filaments at the membrane-cortex interface during the critical initiation step.

cell biology↗

bioRxiv2025

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

Tropomyosin 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. Here, we propose a method in which a hydrodynamic drag force is applied directly to the proteins of interest, by imposing a controlled fluid flow inside a microfluidic chamber. The breaking of the protein bonds is directly visualised with fluorescence microscopy. Using this approach, we reveal that very low forces from 0.01 to 0.1 pN per tropomyosin dimer trigger the detachment of entire tropomyosin clusters from actin filaments. We show that the tropomyosin cluster detachment rate depends on the cytoplasmic tropomyosin isoform (Tpm1.6, 1.7, 1.8) and increases exponentially with the applied force. These observations lead us to propose a cluster detachment model which suggests that tropomyosins dynamically explore different positions over the actin filament. Our experimental setup can be used with many other cytoskeletal proteins, and we show, as a proof-of-concept, that the velocity of myosin-X motors is reduced by an opposing fluid flow. Overall, this method expands the range of protein-protein interactions that can be studied by force spectroscopy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/653996v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@19298b4org.highwire.dtl.DTLVardef@16766beorg.highwire.dtl.DTLVardef@18a70c6org.highwire.dtl.DTLVardef@1f1fad5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Development of Clinically Viable Non-Muscle Myosin II Small Molecule Inhibitors with Broad Therapeutic Potential

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal synaptic plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by the lack of selective tools. The most prototypical non-selective inhibitor, blebbistatin inactivates both NMII and cardiac myosin II (CMII), a key regulator of heart function. Using rational drug design, we developed a series of NMII inhibitors that improve tolerability by selectively targeting NMII over CMII, including MT-228, which has excellent properties such as high brain penetration and efficacy in preclinical models of stimulant use disorder, which has no current FDA-approved therapies. The structure of MT-228 bound to myosin II provides insight into its 17-fold selectivity for NMII over CMII. MT-228s broad therapeutic window opens the door to new disease treatments and provides valuable tools for the scientific community, along with promising leads for future medication development. HighlightsO_LIResearch suggests numerous indications, from axon regeneration and cancer, would benefit from a small molecule inhibitor of non-muscle myosin II, a molecular motor that regulates the actin cytoskeleton. C_LIO_LICurrent chemical probe options are very limited and lack sufficient safety for in vivo C_LIO_LIstudies, which we show is primarily due to potent inhibition of cardiac myosin II. C_LIO_LIRational design that focused on improving target selectivity over the pan-myosin II inhibitor, blebbistatin, led to the identification of MT-228, a small molecule inhibitor with a wide therapeutic window. C_LIO_LIHigh-resolution structure of MT-228 bound to myosin II reveals that selectivity results from a different positioning compared to blebbistatin and an important sequence difference between cardiac and non-muscle myosin II in the inhibitor binding pocket. C_LIO_LIA single administration of MT-228 shows long-lasting efficacy in animal models of stimulant use disorder, a current unmet and rapidly escalating need with no FDA-approved treatments. C_LI

cell biology↗

bioRxiv2024

Ca2+ Regulation of Myosin II and Myosin VI during Rupture of the Shigella-Containing Vacuole

Shigella, the causative agent of bacillary dysentery, invades epithelial cells to colonize the intestinal mucosa. Following invasion, Shigella is enclosed in a vacuole that needs to rupture for bacterial intra-cytosolic replication. We show here that rupture of the Shigella vacuole requires Ca2+ influx leading to long lasting local Ca2+ increases that regulate actin dynamics affecting the Shigella vacuole integrity. These Ca2+ increases promote vacuolar rupture by activating myosin II associated with actin filaments in membrane ruffles distant from the vacuole, while tethering myosin VI at the actin coat-surrounded vacuole. Ca2+ depletion and myosin II inhibition impair formation of the actin coat and vacuole rupture. Inhibition of myosin VI also delays rupture of vacuoles but lead to their tumbling. These findings highlight a role for Ca2+ in coordinating actin-based forces and constraints during early rupture steps of bacterial vacuole, that pull on vacuolar membranes and tether them to the actin cortex via myosin II and VI, respectively, a process relevant to intracellular pathogen and endomembrane trafficking.

microbiology↗

bioRxiv2023

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 {beta}-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 Omecamtiv mecarbil and the inhibitor Mavacamten. In this work, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All atoms molecular dynamics simulations reveal how these molecules can have antagonistic impact on the allostery of the motor by comparing {beta}-cardiac myosin in the apo form or bound to Omecamtiv mecarbil or Mavacamten. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine.

molecular biology↗

bioRxiv2023

A weak coupling mechanism mediates 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. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster that the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is mostly driven by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism. Author summaryMyosin is an ATP-powered motor protein that is crucial for cellular functions like muscle contraction, cell division, and the transport of molecular cargos. Understanding how myosin transforms chemical energy from ATP hydrolysis into mechanical work is a central open question in structural bioenergetics, which could guide the design of next-generation synthetic nanomachines. In myosin, ATP hydrolysis is coupled to the re-priming of the lever-arm during the recovery stroke transition. Using advanced molecular dynamics simulations, we described the sequence of events and the energetics of the recovery stroke of myosin VI with an unprecedented level of detail. The results support a mechanism in which the re-priming of the mechanical amplifier region of the protein is almost entirely driven by thermal fluctuations and stabilized by ATP hydrolysis. This weakly-coupled mechanism suggests that myosin can "rectify" thermal fluctuations to work efficiently in an isothermal environment dominated by stochastic fluctuations like the cell.

biophysics↗

bioRxiv2023

MiniBAR/KIAA0355 is a dual Rac and Rab effector required for ciliogenesis

Cilia protrude from the cell surface and play critical roles in in-tracellular signaling, environmental sensing and development. Actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1-and Rab35-binding protein with a truncated BAR domain that we named MiniBAR (aka KIAA0355/GARRE) which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles traffick-ing to the ciliary base and exhibits remarkable fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia resulting from abnormal Rac-GTP/Rho-GTP levels, increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hall-marks of ciliopathies including left-right asymmetry defects. Thus, MiniBAR is a unique dual Rac and Rab effector that con-trols both actin cytoskeleton and membrane trafficking for cili-ogenesis.

cell biology↗

bioRxiv2023

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, proper functioning of Myo6 relies on precise spatio-temporal 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. TOM1 and Dab2 cannot bind the off-state while, GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

biochemistry↗

bioRxiv2023

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. The off-state is equated with a folded-back structure known as the interacting head motif (IHM), which is a regulatory feature of all muscle myosins and class-2 non-muscle myosins. We report here the human {beta}-cardiac myosin IHM structure to 3.6 [A] resolution. The structure shows that the interfaces are hot spots of HCM mutations and reveals details of the significant interactions. Importantly, the structures of cardiac and smooth muscle myosin IHMs are dramatically different. This challenges the concept that the IHM structure is conserved in all muscle types and opens new perspectives in the understanding of muscle physiology. The cardiac IHM structure has been the missing puzzle piece to fully understand the development of inherited cardiomyopathies. This work will pave the way for the development of new molecules able to stabilize or destabilize the IHM in a personalized medicine approach. *This manuscript was submitted to Nature Communications in August 2022 and dealt efficiently by the editors. All reviewers received this version of the manuscript before 9208 August 2022. They also received coordinates and maps of our high resolution structure on the 18208 August 2022. Due to slowness of at least one reviewer, this contribution was delayed for acceptance by Nature Communications and we are now depositing in bioRxiv the originally submitted version written in July 2022 for everyone to see. Indeed, two bioRxiv contributions at lower resolution but adding similar concepts on thick filament regulation were deposited this week in bioRxiv, one of the contributions having had access to our coordinates. We hope that our data at high resolution will be helpful for all readers that appreciate that high resolution information is required to build accurate atomic models and discuss implications for sarcomere regulation and the effects of cardiomyopathy mutations on heart muscle function.

biophysics↗

bioRxiv2022

Nucleotide-free structures of Kif20A illuminate the atypical allostery in this mitotic 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. We present here the first high-resolution structure of its motor domain that delineates the highly unusual structural features of this motor, including a long L6 insertion that integrates into the core of the motor domain and that drastically affects allostery and ATPase activity. Together with the high-resolution cryo-EM microtubule-bound KIF20A structure that reveal the microtubule-binding interface, we dissect the peculiarities of the KIF20A sequence that work to favor fast dissociation of ADP, particularly in contrast to other kinesins. Structural and functional insights from the KIF20A pre-power stroke conformation thus highlight the role of extended insertions in shaping the motor mechanochemical cycle. Essential for force production and processivity is the length of the neck linker in kinesins. We highlight here the role of the sequence preceding the neck linker in controlling its backward docking and show that a neck linker 4-times longer than kinesin-1 is required for the activity of this motor.

biochemistry↗

bioRxiv2022

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

Malaria is responsible for more than a half million deaths per year. The Plasmodium parasites responsible continue to develop resistance to all known agents, despite treatment with different antimalarial combinations. The atypical Myosin A 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. KNX-002 inhibits PfMyoA ATPase activity in vitro and blocks asexual blood stage growth of merozoites, one of three motile Plasmodium life-cycle stages. Combining biochemical assays, X-ray crystallography and molecular dynamics, we demonstrate that KNX-002 targets a novel pocket in PfMyoA, sequestering it in a post-rigor state detached from actin. KNX-002 binding affects Mg2+ coordination near ATP, preventing ATP hydrolysis and thus inhibiting motor activity. This first-in-class small-molecule inhibitor of PfMyoA paves the way for developing a new generation of antimalarial treatments.

molecular biology↗

bioRxiv2021

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 is 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. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

biochemistry↗

bioRxiv2021

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. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin binding protein, the state of the actin cytoskeleton and MF myosin activity.

cell biology↗

bioRxiv2021

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 S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

cell biology↗

bioRxiv2020

Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity and ultimately pathogenesis of this parasite relies on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future antimalarials targeting both the glideosome motor and its regulatory elements.HighlightsThe first structures of the full length PfMyoA motor in two states of its motor cycle.A unique priming of the PfMyoA lever arm results from specific lever arm/motor domain interactions, which allows for a larger powerstroke to enhance speed.Sequence adaptations within the motor domain and degenerate IQ motifs in the lever arm dictate PfMyoA motor properties.PfELC is essential for blood cell invasion and is a weak link in the assembly of a fully functional motor, providing a second novel target for antimalarial drug design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2018

Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition

AbstractHypertrophic cardiomyopathies (HCM) result from distinct single-point mutations in sarcomeric proteins that lead to muscle hypercontractility. While different models account for a pathological increase in the power output, clear understanding of the molecular basis of dysfunction in HCM is the mandatory next step to improve current treatments. An optimized quasi-atomic model of the sequestered state of cardiac myosin coupled to X-ray crystallography and in silico analysis of the mechanical compliance of the lever arm allowed us to perform a systematic study for a large set of HCM mutations. We define different classes of mutations depending on their effects on lever arm compliance, sequestered state stability and motor functions. This work reconciles previous models and explains how distinct HCM mutations can have disparate effects on the motor mechano-chemical parameters and yet lead to the same disease. The framework presented here can guide future investigations aiming at finding HCM treatments.

biophysics↗