Discovery of novel cardiac troponin activators using fluorescence polarization-based high throughput screening assays.

The large unmet demand for new heart failure therapeutics is widely acknowledged. Over the last decades the contractile myofilaments themselves have emerged as an attractive target for the development of new therapeutics for both systolic and diastolic heart failure. However, the clinical use of myofilament-directed drugs has been limited, and further progress has been hampered by incomplete understanding of myofilament function on the molecular level and screening technologies for small molecules that accurately reproduce this function in vitro.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled).

The Crystal Structure of Pneumolysin at 2.0 Å Resolution Reveals the Molecular Packing of the Pre-pore Complex.

Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available.

Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome.

Background: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure.

Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting.

Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1.

Tropomyosin dynamics.

Tropomyosin is a two chained α-helical coiled coil protein that binds actin filaments and interacts with various actin binding proteins. Tropomyosin function depends on its ability to move to distinct locations on the surface of actin in response to the binding of different thin filament effectors. Tropomyosin dynamics plays an important role in these fluctuating interactions with actin and is thought to be fundamental to many of its biological activities.

Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients.

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca(2+) sensitivity.

Effects of basic calponin on the flexural mechanics and stability of F-actin.

The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. In this work, we studied the mechanical and structural interaction of actin with basic calponin, a differentiation marker in smooth muscle cells, on a single filament level.

A structural and functional dissection of the cardiac stress response factor MS1.

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F-actin binding ability, located to the C-terminal half of the protein, which promotes stabilization of F-actin in the cell thus releasing myocardin-related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor.

The calponin regulatory region is intrinsically unstructured: novel insight into actin-calponin and calmodulin-calponin interfaces using NMR spectroscopy.

Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131-228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution.

Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments: evidence for a cooperative switching mechanism.

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca(2+)-binding protein and their interaction with myosin is Ca(2+)-regulated. We suggested that Ca(2+) regulation by caldesmon and Ca(2+)-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis.

The effect of mutations in alpha-tropomyosin (E40K and E54K) that cause familial dilated cardiomyopathy on the regulatory mechanism of cardiac muscle thin filaments.

E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca(2+) sensitivity. E40K also reduces V(max) of actin-Tm-activated S-1 ATPase by 18%.

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase.

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%.

Cooperative inhibition of actin filaments in the absence of tropomyosin.

We measured the inhibition of actin activated myosin subfragment-1 MgATPase activity in a solution containing no added KCl (5 mM PIPES.K2 (pH 7.1), 2.

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties.

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin.

The kinetic mechanism of Myo1e (human myosin-IC).

Myo1e is the widely expressed subclass-1 member of the myosin-I family. We performed a kinetic analysis of a truncated myo1e that consists of the motor and the single IQ motif with a bound calmodulin. We determined the rates and equilibrium constants for the key steps in the ATPase cycle.