Biology subjects

Spudich, J.

Publications and source records attributed to Spudich, J..

8 recordsLinked to original sources

bioRxiv2026

The hypertrophic cardiomyopathy mutation G768R makes cardiac myosin a high duty ratio motor

{beta}-cardiac myosin is the primary motor protein in the human heart responsible for force generation by converting chemical energy from ATP hydrolysis to mechanical work. It binds actin, produces force through its powerstroke, and releases actin, and must complete this cycle several times a second within each heartbeat. Like other muscle myosins, cardiac myosin has a low duty ratio of [~]5%, the fraction of time in the actin-bound force-producing state. Here we present a hypertrophic cardiomyopathy (HCM) causing mutation, G768R, that increases cardiac myosins duty ratio to >60%, an unprecedented >10x increase unmatched by any previously studied muscle myosin mutation. In recombinantly expressed human {beta}-cardiac myosin subfragment-1 (sS1), G768R dramatically decreases the load-sensitive actin-detachment rate and step size of single molecules measured by optical tweezers. The [~]15x longer actin-bound time combined with only a modest change in overall ATPase rate predicts a duty ratio of >60%. Motility velocity is also severely slowed, as expected given the long bound time and decreased step size. All-atom molecular dynamic simulations of the pre-powerstroke and post-rigor states predict that the mutation alters lever arm priming and reduces ADP pocket opening, providing a possible structural mechanism to corroborate the experimental observations. Finally, ATPase experiments on 2-headed heavy meromyosin (HMM) constructs suggest that G768R destabilizes the autoinhibited state of myosin. Our findings of high duty ratio and reduced autoinhibition provide molecular mechanisms of cardiac hypercontractility and impaired relaxation. A muscle myosin in the heart that stays bound to actin for over half its period is bound to have significant functional and clinical consequences.

biophysics↗

bioRxiv2024

A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human {beta}-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

biophysics↗

bioRxiv2024

Broad therapeutic benefit of myosin inhibition in hypertrophic cardiomyopathy

Myosin inhibitor mavacamten is the only targeted treatment available for hypertrophic cardiomyopathy (HCM), a disease caused by hundreds of genetic variants that affect mainly sarcomeric myosin and its negative regulator cardiac myosin-binding protein C (cMyBP-C, encoded by MYBPC3). Here, we have examined whether the reported limited efficacy of mavacamten in a fraction of HCM patients can result from dissimilar HCM pathomechanisms triggered by different genetic variants, a scenario particularly relevant for MYBPC3-associated HCM. To this aim, we have generated knock-in mice including missense pathogenic variant cMyBP-C p.R502W, which, different from patients who carry truncations in the protein, develop progressive pathogenic myocardial remodeling in the absence of alterations of cMyBP-C levels and localization. Mechanistically, we find that mutation R502W reduces the binding affinity of cMyBP-C for myosin without inducing a shift towards more active myosin conformations as observed when cMyBP-C levels are reduced. Despite these diverging molecular alterations, we show that mavacamten blunts myocardial remodeling both in R502W and cMyBP-C-deficient, knock-out hearts. These beneficial effects are accompanied by improved tolerance to exercise only in R502W animals. Hence, our results indicate that myosin inhibition is effective to treat HCM caused by both truncating and missense variants in MYBPC3 regardless of the primary pathomechanisms they elicit.

physiology↗

bioRxiv2022

A small-molecule myosin inhibitor as a targeted multi-stage antimalarial

Malaria is a devastating disease that resulted in an estimated 627,000 deaths in 2020. About 80% of those deaths were among children under the age of five. Our approach is to develop small molecule inhibitors against cytoskeletal targets that are vital components of parasite function, essential at multiple stages of parasite infection, can be targeted with high specificity, and are highly druggable. Here we describe KNX-115, which inhibits purified Plasmodium falciparum myosin A (PfMyoA) actin-activated ATPase with a potency in the 10s of nanomolar range and >50-fold selectivity against cardiac, skeletal, and smooth muscle myosins. KNX-115 inhibits the blood and liver stages of Plasmodium with an EC50 of about 100 nanomolar, with negligible liver cell toxicity. In addition, KNX-115 inhibits sporozoite cell traversal and blocks the gametocyte to oocyst conversion in the mosquito. KNX-115 displays a similar killing profile to pyrimethamine and parasites are totally killed after 96 hours of treatment. In line with its novel mechanism of action, KNX-115 is equally effective at inhibiting a panel of Plasmodium strains resistant to experimental and marketed antimalarials. In vitro evolution data likely suggests a refractory potential of KNX-115 in developing parasite resistance.

pharmacology and toxicology↗

bioRxiv2021

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function

Mutations in the lever arm of {beta}-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy (HCM), a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin S2 tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of {beta}-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

biochemistry↗

bioRxiv2019

A novel role for Myosin-Va in mitochondrial fission

In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC=\"FIGDIR/small/655803v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (35K):\norg.highwire.dtl.DTLVardef@1130e72org.highwire.dtl.DTLVardef@196619org.highwire.dtl.DTLVardef@17e09edorg.highwire.dtl.DTLVardef@1bdc311_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Crystal Structure of a Natural Light-Gated Anion Channelrhodopsin

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 [A] reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

biochemistry↗

bioRxiv2018

Controlling load-dependent contractility of the heart at the single molecule level

Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate from actin of myosin, which determines the time that myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which myosin works. Here, we measure the detachment rate of single molecules of human {beta}-cardiac myosin and its load dependence. We find that both can be modulated by both small molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating vs. inhibitory perturbations of cardiac myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and that cardiac contractility can be controlled by tuning the load-dependent kinetics of single myosin molecules.

biophysics↗