CryoEM structure determination of actin decorated with smooth muscle heavy meromyosin
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ABSTRACT:
SUBMITTER: Alimohammad Hojjatian
PROVIDER: EMPIAR-11555 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.
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Project description:Smooth muscle myosin is activated by regulatory light chain (RLC) phosphorylation. In the unphosphorylated state the activity of both heads is suppressed due to an asymmetric, intramolecular interaction between the heads. The properties of myosin with only one of its two RLCs phosphorylated, a state likely to be present both during the activation and the relaxation phase of smooth muscle, is less certain despite much investigation. Here we further characterize the mechanical properties of an expressed heavy meromyosin (HMM) construct with only one of its RLCs phosphorylated (HMM-1P). This construct was previously shown to have more than 50% of the ATPase activity of fully phosphorylated myosin (HMM-2P) and to move actin at the same speed in a motility assay as HMM-2P (Rovner, A. S., Fagnant, P. M., and Trybus, K. M. (2006) Biochemistry 45, 5280-5289). Here we show that the unitary step size and attachment time to actin of HMM-1P is indistinguishable from that of HMM-2P. Force-velocity measurements on small ensembles show that HMM-1P can generate approximately half the force of HMM-2P, which may relate to the observed duty ratio of HMM-1P being approximately half that of HMM-2P. Therefore, single-phosphorylated smooth muscle HMM molecules are active species, and the head associated with the unphosphorylated RLC is mechanically competent, allowing it to make a substantial contribution to both motion and force generation during smooth muscle contraction.
| S-EPMC2709353 | biostudies-literature
Project description:Regulation of the actin-activated ATPase of smooth muscle myosin II is known to involve an interaction between the two heads that is controlled by phosphorylation of the regulatory light chain. However, the three-dimensional structure of this inactivated form has been unknown. We have used a lipid monolayer to obtain two-dimensional crystalline arrays of the unphosphorylated inactive form of smooth muscle heavy meromyosin suitable for structural studies by electron cryomicroscopy of unstained, frozen-hydrated specimens. The three-dimensional structure reveals an asymmetric interaction between the two myosin heads. The ATPase activity of one head is sterically "blocked" because part of its actin-binding interface is positioned onto the converter domain of the second head. ATPase activity of the second head, which can bind actin, appears to be inhibited through stabilization of converter domain movements needed to release phosphate and achieve strong actin binding. When the subfragment 2 domain of heavy meromyosin is oriented as it would be in an actomyosin filament lattice, the position of the heads is very different from that needed to bind actin, suggesting an additional contribution to ATPase inhibition in situ.
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Project description:Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.
| S-EPMC10710074 | biostudies-literature
Project description:Recent advances in cryo-electron microscopy (cryoEM) have dramatically improved the resolutions at which vitrified biological specimens can be studied, revealing new structural and mechanistic insights over a broad range of spatial scales. Bolstered by these advances, much effort has been directed toward the development of hybrid modeling methodologies for the construction and refinement of high-fidelity atomistic models from cryoEM data. In this brief review, we will survey the key elements of cryoEM-based hybrid modeling, providing an overview of available computational tools and strategies as well as several recent applications.
| S-EPMC5934336 | biostudies-literature
Project description:Missense variants throughout ACTA2, encoding smooth muscle α-actin (αSMA), predispose to adult-onset thoracic aortic disease, but variants disrupting arginine 179 (R179) lead to Smooth Muscle Dysfunction Syndrome (SMDS) characterized by diverse childhood-onset vascular diseases. Here we show that αSMA localizes to the nucleus in wildtype (WT) smooth muscle cells (SMCs), enriches in the nucleus with SMC differentiation, and associates with chromatin remodeling complexes and SMC contractile gene promotors. The ACTA2 p.R179 αSMA variant shows decreased nuclear localization. Primary SMCs from Acta2 SMC-R179C/+ mice are less differentiated than WT SMCs in vitro and in vivo and have global changes in chromatin accessibility. Induced pluripotent stem cells from patients with ACTA2 p.R179 variants fail to fully differentiate from neuroectodermal progenitor cells to SMCs, and single-cell transcriptomic analyses of an ACTA2 p.R179H patient's aortic tissue show increased SMC plasticity. Thus, nuclear αSMA participates in SMC differentiation, and loss of this nuclear activity occurs with ACTA2 p.R179 pathogenic variants.
| S-EPMC11198982 | biostudies-literature
Project description:The binding of the Ca2+-regulated native thin filaments from vascular smooth muscle to vascular smooth-muscle heavy meromyosin was measured in the presence of 3 mM-MgATP. At 25 degrees C and I 0.25 binding had an affinity of 1 X 10(-6)-0.3 X 10(-6) M-1 with a stoichiometry of one molecule bound to one actin monomer. The Km for the activation of heavy-meromyosin ATPase was 20-50 microM. Thin filament-heavy meromyosin binding was not altered by Ca2+ (pCa 9-4) or the extent of myosin phosphorylation. With skeletal-muscle heavy meromyosin affinity was 0.023 X 10(6) M-1 in parallel with activation of the ATPase (Km 54 microM). It is concluded that tight binding is specific to smooth-muscle proteins and that it is not related to the ATPase activation site.
| S-EPMC1138506 | biostudies-other
Project description:Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.
| S-EPMC5748330 | biostudies-literature
Project description:Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.
| S-EPMC4866761 | biostudies-literature
Project description:AimsThe variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis.Methods and resultsApoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice.ConclusionThese data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.
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