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I_2020_BPJ_Sundar_Moore_A277V_Tpm.pdf (1.2 MB)

Cardiomyopathy mutation alters end-to-end junction of tropomyosin and reduced calcium sensitivity

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posted on 2021-03-16, 02:30 authored by SaiLavanyaa Sundar, Michael J. Rynkiewicz, Anita Ghosh, William Lehman, Jeffrey R Moore
Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (Blocked, Closed and Open) in response to calcium-binding to troponin and acto-myosin cross-bridge formation. The Tpm coiled coil polymerizes head-to-tail along the long-pitch helix of F-actin to form continuous super-helical cables that wrap around the actin filaments. The end-to-end bonds formed between the N- and C- terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter tropomyosin binding and thin filament activation by altering the overlap structure. Here, we used co-sedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin, and its ability to regulate acto-myosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin - Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in tropomyosin flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca2+ sensitivity, consistent with a mutation-induced bolstering of B-state Tpm-actin electrostatic contacts and an increased TnT1 binding affinity, was also observed.

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20PRE35120307

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