posted on 2021-03-16, 02:30authored bySaiLavanyaa 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.