Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca2+ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament.

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca2+ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.

Ca2+ attenuates nucleation activity of leiomodin.

A transient increase in Ca2+ concentration in sarcomeres is essential for their proper function. Ca2+ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin-binding protein C were also found to be Ca2+ sensitive. However, the effects of Ca2+ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene-actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca2+ . Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin-binding sites binds Ca2+ . We propose that Ca2+ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca2+ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function. SUMMARY STATEMENT: Ca2+ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca2+ on the activity of leiomodin-an actin nucleator and thin filament length regulator-have remained unknown. In this study, we demonstrate that Ca2+ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca2+ in the regulation of the sarcomeric protein interactions.

Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.

The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.

Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin.

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels. Under activating conditions Ca2+ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca2+, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca2+ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament.

Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective action is poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD, which is absent in skeletal isoforms of MyBP-C, and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.

Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy.

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca2+ The current model of muscle regulation holds that at relaxing (low-Ca2+) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca2+) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca2+, tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca2+ and lead to a mechanistic model for the regulation of the cardiac TF.

Filaments and fingers: Novel structural aspects of the single septin from Chlamydomonas reinhardtii.

Septins are filament-forming GTP-binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self-assemble into heterocomplexes, which polymerize into highly organized, cell membrane-interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms. This makes the single septin from Chlamydomonas (CrSEPT) a particularly attractive model for investigating whether functional homopolymeric septin filaments also exist. CrSEPT was detected at the base of the flagella in Chlamydomonas, suggesting that CrSEPT is involved in the formation of a membrane-diffusion barrier. Using transmission electron microscopy, we observed that recombinant CrSEPT forms long filaments with dimensions comparable with those of the canonical structure described for opisthokonts. The GTP-binding domain of CrSEPT purified as a nucleotide-free monomer that hydrolyzes GTP and readily binds its analog guanosine 5'-3-O-(thio)triphosphate. We also found that upon nucleotide binding, CrSEPT formed dimers that were stabilized by an interface involving the ligand (G-interface). Across this interface, one monomer supplied a catalytic arginine to the opposing subunit, greatly accelerating the rate of GTP hydrolysis. This is the first report of an arginine finger observed in a septin and suggests that CrSEPT may act as its own GTP-activating protein. The finger is conserved in all algal septin sequences, suggesting a possible correlation between the ability to form homopolymeric filaments and the accelerated rate of hydrolysis that it provides.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. Here, we show that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, we determine that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). We show that each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, we show that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, our structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis.