Cardiomyopathy Mutation Alters End-to-End Junction of Tropomyosin and Reduces Calcium Sensitivity

Abstract

Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (B-, C-, and M-states) in response to calcium binding to troponin and actomyosin cross-bridge formation. The Tpm coiled coil polymerizes head to tail along the long-pitch helix of F-actin to form continuous superhelical 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 Tpm binding and thin filament activation by altering the overlap structure. Here, we used cosedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin and its ability to regulate actomyosin 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 Tpm flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca 2þ sensitivity, consistent with a mutationinduced bolstering of the B-state Tpm-actin electrostatic contacts and an increased Tpm troponin T1 binding affinity, was also observed.

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end

“…7) is consistent with decreased strength of the tropomyosin end-to-end bond (30). It is important to note, however, that although often considered a direct hallmark of cooperativity, tropomyosintropomyosin affinity does not necessarily correlate with cooperativity (5). Rather, it appears that end-to-end bond mechanical rigidity (5), not affinity, indicates the ability for tropomyosin position to be transmitted between cooperative units.…”

“…To further elucidate why the troponin T N terminus fragment failed to affect the M8R-tropomyosin-regulated actin in the in vitro motility assay, the ability of troponin T1 to actin filaments with bound mutant M8R-and WT-tropomyosin was determined via co-sedimentation. At saturating concentration for troponin T1 fragment binding to WT tropomyosin (5,12), the presence of M8R tropomyosin dramatically diminished this interaction (Fig. 6B).…”

“…All co-sedimentations were performed in triplicate and in sample buffer containing 15 mM BES, 5 mM MgCl 2 , 55 mM KCl, at pH 7.0, and carried out as described previously (5). Briefly, the samples were incubated at room temperature for 20 min, followed by centrifugation at 130,000 3 g for 20 min at 4°C.…”

“…Molecular dynamics and analysis of tropomyosin end-to-end overlap domain Molecular dynamics simulations were performed on the overlap domain of tropomyosin as described previously (5,15). All simulations were performed in nanoscale molecular dynamics (45) in explicit solvent at 300 K and 1 atm.…”

“…Tropomyosin molecules twist together as a parallel dimer that forms an a-helical coiled-coil. The tightly wound nature of tropomyosin provides little protection from local mutationinduced structural changes having long-range effects (4,5) that can act a hundred or more angstroms away from the site of the mutation (4,6). Each tropomyosin dimer binds seven consecutive actin subunits and, when bound end-to-end with adjacent tropomyosin dimers, creates a continuous cable that wraps around the actin filament.…”