New paradigms in actomyosin energy transduction: Critical evaluation of non-traditional models for orthophosphate release.

Release of the ATP hydrolysis product ortophosphate (Pi) from the active site of myosin is central in chemo-mechanical energy transduction and closely associated with the main force-generating structural change, the power-stroke. Despite intense investigations, the relative timing between Pi-release and the power-stroke remains poorly understood. This hampers in depth understanding of force production by myosin in health and disease and our understanding of myosin-active drugs. Since the 1990s and up to today, models that incorporate the Pi-release either distinctly before or after the power-stroke, in unbranched kinetic schemes, have dominated the literature. However, in recent years, alternative models have emerged to explain apparently contradictory findings. Here, we first compare and critically analyze three influential alternative models proposed previously. These are either characterized by a branched kinetic scheme or by partial uncoupling of Pi-release and the power-stroke. Finally, we suggest critical tests of the models aiming for a unified picture.

Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy.

Hereditary hypertrophic cardiomyopathy (HCM), due to mutations in sarcomere proteins, occurs in more than 1/500 individuals and is the leading cause of sudden cardiac death in young people. The clinical course exhibits appreciable variability. However, typically, heart morphology and function are normal at birth, with pathological remodeling developing over years to decades, leading to a phenotype characterized by asymmetric ventricular hypertrophy, scattered fibrosis and myofibrillar/cellular disarray with ultimate mechanical heart failure and/or severe arrhythmias. The identity of the primary mutation-induced changes in sarcomere function and how they trigger debilitating remodeling are poorly understood. Support for the importance of mutation-induced hypercontractility, e.g., increased calcium sensitivity and/or increased power output, has been strengthened in recent years. However, other ideas that mutation-induced hypocontractility or non-uniformities with contractile instabilities, instead, constitute primary triggers cannot yet be discarded. Here, we review evidence for and criticism against the mentioned hypotheses. In this process, we find support for previous ideas that inefficient energy usage and a blunted Frank-Starling mechanism have central roles in pathogenesis, although presumably representing effects secondary to the primary mutation-induced changes. While first trying to reconcile apparently diverging evidence for the different hypotheses in one unified model, we also identify key remaining questions and suggest how experimental systems that are built around isolated primarily expressed proteins could be useful.

Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?

In muscle, but not in single-molecule mechanics studies, actin, myosin and accessory proteins are incorporated into a highly ordered myofilament lattice. In view of this difference we compare results from single-molecule studies and muscle mechanics and analyze to what degree data from the two types of studies agree with each other. There is reasonable correspondence in estimates of the cross-bridge power-stroke distance (7⁻13 nm), cross-bridge stiffness (~2 pN/nm) and average isometric force per cross-bridge (6⁻9 pN). Furthermore, models defined on the basis of single-molecule mechanics and solution biochemistry give good fits to experimental data from muscle. This suggests that the ordered myofilament lattice, accessory proteins and emergent effects of the sarcomere organization have only minor modulatory roles. However, such factors may be of greater importance under e.g., disease conditions. We also identify areas where single-molecule and muscle data are conflicting: (1) whether force generation is an Eyring or Kramers process with just one major power-stroke or several sub-strokes; (2) whether the myofilaments and the cross-bridges have Hookean or non-linear elasticity; (3) if individual myosin heads slip between actin sites under certain conditions, e.g., in lengthening; or (4) if the two heads of myosin cooperate.

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays.

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5-1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8-9 s-1) and actin concentration for half maximal activity (KATPase; 70-80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Multistep orthophosphate release tunes actomyosin energy transduction.

Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms.

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s-1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold.

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

Dissecting how modular polyketide synthase ketoreductases interact with acyl carrier protein-attached substrates.

Interaction studies using fragments excised from the modular mycolactone polyketide synthase show that ketoreductase domains possess a generic binding site for acyl carrier protein domains and provide evidence that the pendant 5'-phosphopantetheine prosthetic group plays a key role in delivering acyl substrates to the active site in the correct orientation.

Portability and Structure of the Four-Helix Bundle Docking Domains of trans-Acyltransferase Modular Polyketide Synthases.

The polypeptides of multimodular polyketide synthases self-assemble into biosynthetic factories. While the docking domains that mediate the assembly of cis-acyltransferase polyketide synthase polypeptides are well-studied, those of the more recently discovered trans-acyltransferase polyketide synthases have just started to be described. Located at the C- and N-termini of many polypeptides, these 25-residue, two-helix, pseudosymmetric motifs noncovalently connect domains both between and within modules. Domains expressed with their natural, cognate docking motifs formed complexes stable to size-exclusion chromatography with 1-10 µM dissociation constants as measured by isothermal titration calorimetry. Deletion and swapping experiments demonstrate portability of the docking motifs. A 1.72 Å-resolution structure of the N-terminal portion of the macrolactin synthase polypeptide MlnE shows an uncomplexed N-terminal docking motif to be preorganized in the conformation it assumes within the docking domain complex.