The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium.

Human androgen receptor contains a large N-terminal domain (AR-NTD) that is highly dynamic and this poses a major challenge for experimental and computational analysis to decipher its conformation. Misfolding of the AR-NTD is implicated in prostate cancer and Kennedy's disease, yet our knowledge of its structure is limited to primary sequence information of the chain and a few functionally important secondary structure motifs. Here, we employed an innovative combination of molecular dynamics simulations and circuit topology (CT) analysis to identify the tertiary structure of AR-NTD. We found that the AR-NTD adopts highly dynamic loopy conformations with two identifiable regions with distinct topological make-up and dynamics. This consists of a N-terminal region (NR, residues 1-224) and a C-terminal region (CR, residues 225-538), which carries a dense core. Topological mapping of the dynamics reveals a traceable time-scale dependent topological evolution. NR adopts different positioning with respect to the CR and forms a cleft that can partly enclose the hormone-bound ligand-binding domain (LBD) of the androgen receptor. Furthermore, our data suggest a model in which dynamic NR and CR compete for binding to the DNA-binding domain of the receptor, thereby regulating the accessibility of its DNA-binding site. Our approach allowed for the identification of a previously unknown regulatory binding site within the CR core, revealing the structural mechanisms of action of AR inhibitor EPI-001, and paving the way for other drug discovery applications. K E Y W O R D Sandrogen receptor, circuit topology, conformational dynamics, intrachain contacts, nuclear hormone receptors Vahid Sheikhhassani and Barbara Scalvini contributed equally to this study.

Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment.

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