Troponin C (TnC) facilitates muscle contraction through calcium-binding within its N-terminal region (NTnC). As previously observed using molecular dynamics (MD) simulations, this calcium-binding event leads to an increase in the dynamics of helices lining a hydrophobic patch on TnC. Simulation times of multiple microseconds were required to even see a partial opening of the hydrophobic patch, limiting the ability to thoroughly and quantitatively investigate these rare events. Here we describe the application of umbrella sampling to probe the TnC hydrophobic patch opening in a more targeted and quantitative fashion. Umbrella sampling was utilized to investigate the differences in the free energy of opening between cardiac (cTnC) and fast skeletal TnC (sTnC). We found that, in agreement with previous reports, holo (calcium-bound) sTnC had a lower free energy of opening compared with holo cTnC. Additionally, differences in the free energy of opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. MD simulations and umbrella sampling revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. An umbrella sampling simulation of cTnI-bound holo cTnC exhibited the lowest free energy in the open configuration, in agreement with experimental data. In conclusion, this study presents a novel and successful protocol for applying umbrella sampling simulations to quantitatively study the molecular basis of muscle contraction and proposes a mechanism by which HCM and DCM-associated mutations influence contraction.

Troponin is a key regulatory protein in muscle contraction, consisting of three subunits troponin C (TnC), troponin I (TnI), and troponin T (TnT). Calcium association to TnC initiates contraction by causing a series of dynamic and conformational changes that allow the switch peptide of TnI to bind and subsequently cross bridges to form between the thin and thick filament of the sarcomere. Owing to its pivotal role in contraction regulation, troponin has been the focus of numerous computational studies over the last decade. These studies elegantly supplemented a large volume of experimental work and focused on the structure, dynamics and function of the whole troponin complex, individual subunits, and even on segments of the thin filament. Molecular dynamics, Brownian dynamics, and free energy simulations have been used to elucidate the conformational dynamics and underlying free energy landscape of troponin, calcium, and switch peptide binding, as well as the effect of disease mutations, small molecules and post-translational modifications such as phosphorylation. Frequently, simulations have been used to confirm or explain experimental observations. Computer-aided drug discovery tools have been employed to identify novel potential calcium sensitizing agents binding to the TnC-TnI interface. Finally, Markov modeling has contributed to simulating contraction within the sarcomere on the mesoscale. Here we are reviewing and classifying the existing computational work on troponin and its subunits, outline current gaps in simulations elucidating troponin's role in contraction and suggest potential future developments in the field.

Cardiac troponin C (cTnC) binds intracellular calcium and subsequently cardiac troponin I (cTnI), initiating cardiac muscle contraction. Due to its role in contraction, cTnC has been a therapeutic target in the search for small molecules to treat conditions that interfere with normal muscle contraction like the heritable cardiomyopathies. Structural studies have shown the binding location of small molecules such as bepridil, dfbp-o, 3-methyldiphenylamine (DPA) and W7 to be a hydrophobic pocket in the regulatory domain of cTnC (cNTnC) but have not shown the influence of these small molecules on the energetics of opening this domain. Here we describe an application of an umbrella sampling method used to elucidate the impact these calcium sensitivity modulators have on the free energy of cNTnC hydrophobic patch opening. We found that all these molecules lowered the free energy of opening in the absence of the cTnI, with bepridil facilitating the least endergonic transformation. In the presence of cTnI, however, we saw a stabilization of the open configuration due to DPA and dfbp-o binding, and a destabilization of the open configuration imparted by bepridil and W7. Predicted poor binding molecule NSC34337 left the hydrophobic in under 3 ns in conventional MD simulations suggesting that only hydrophobic patch binders stabilized the open conformation. In conclusion, this study presents a novel approach to study the impact of small molecules on hydrophobic patch opening through umbrella sampling and proposes mechanisms for calcium sensitivity modulation.

Humans are unique in their sophisticated culture and societal structures, their complex languages, and their extensive tool use. According to the human self-domestication hypothesis, this unique set of traits may be the result of an evolutionary process of self-induced domestication, in which humans evolved to be less aggressive and more cooperative. However, the only other species that has been argued to be self-domesticated besides humans so far is bonobos, resulting in a narrow scope for investigating this theory limited to the primate order. Here, we propose an animal model for studying self-domestication: the elephant. First, we support our hypothesis with an extensive cross-species comparison, which suggests that elephants indeed exhibit many of the features associated with self-domestication (e.g., reduced aggression, increased prosociality, extended juvenile period, increased playfulness, socially regulated cortisol levels, and complex vocal behavior). Next, we present genetic evidence to reinforce our proposal, showing that genes positively selected in elephants are enriched in pathways associated with domestication traits and include several candidate genes previously associated with domestication. We also discuss several explanations for what may have triggered a self-domestication process in the elephant lineage. Our findings support the idea that elephants, like humans and bonobos, may be self-domesticated. Since the most recent common ancestor of humans and elephants is likely the most recent common ancestor of all placental mammals, our findings have important implications for convergent evolution beyond the primate taxa, and constitute an important advance toward understanding how and why self-domestication shaped humans’ unique cultural niche.