Impact of sequence changes on key intrinsically disordered regions of cardiac troponin

/Abstract Cardiac contraction is a tightly regulated process, where the heart needs to generate sufficient power to perfuse the body during systole and then relax to allow for filling of the ventricles during diastole. Disruption of this regulation can lead to diseases including cardiomyopathy and heart failure. At the molecular scale, cardiac muscle contraction is powered by myosin molecular motors pulling on thin filaments consisting of actin, troponin, and tropomyosin. The troponin complex, comprised of troponins I, T, and C, plays a central role in regulating contraction by positioning tropomyosin to enable or disable the calcium-dependent interactions between myosin and thin filaments. Altering the sequence composition of troponin subunits has direct effects on its function, and point mutations in troponin are prominent causes of cardiomyopathies; however, it is still a major challenge for the field to connect how changes in sequence affect troponin's functio n. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of the troponin complex; however, there are large, unresolved segments of troponin, which include critical phosphorylation sites and the locations of many pathogenic mutations. These unresolved yet functionally-significant regions likely contain intrinsically disordered regions (IDRs) with behaviors that are not well described by traditional structural approaches. Advances in the study of protein disorder have revealed new paradigms connecting sequence, disorder, and function, and it clear that sequence changes in IDRs could impact function by disrupting binding motifs, altering IDR dynamics, tuning conformational biases, introducing new aberrant interaction sites, and/or affecting interactions with binding partners. Our central hypothesis is that mutations and key post-translational modifications within troponin's IDRs alter: (1) intramolecular interactions, (2) intermolecular interactions, or (3) both. Changes in intramolecular interactions will alter intrinsic IDR conformational biases and/or dynamics, while changes in intermolecular interactions will alter interactions between troponin subunits and/or the well defined troponin binding partners: calcium, tropomyosin, actin, myosin. Here, we have assembled a team with expertise in the sequence and simulation of IDRs (Holehouse), single-molecule structural dynamics of IDRs (Soranno), and cardiac troponin (Greenberg) that will apply cutting-edge experimental and computational tools to study the connections between sequence, disorder, and function in key IDRs within troponin. Together, we will test the following specific hypotheses: (1) Regions predicted to be disordered within troponins T and I are indeed disordered in isolation and in the context of the thin filament. (2) Both phosphorylation and pathogenic mutations within troponin IDRs lead to interpretable/predictable changes in the underlying structural ensemble, conformational dynamics, and/or intermolecular interactions with well-established binding partners, which in turn alter thin filament activation. (3) Applying integrative tools across scales will enable us to better predict whether key VUS within IDRs are likely pathogenic or benign. These studies will provide new insights into the biophysical properties of IDRs and the structure-function relationship in troponin. Moreover, these studies have the potential to aid in the classification of newly identified variants of unknown significance found in patients with cardiomyopathy. . Project Number: 1R01HL179124-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Michael Greenberg (+2 co-PIs) | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $776,791 | Activity Code: R01 | Study Section: Macromolecular Structure and Function C Study Section[MSFC] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17912401