Myopathic mutations in the ß-chain of tropomyosin differently affect the structural and functional properties of ßß- and αß-dimers.

Tropomyosin (Tpm) is an actin-binding protein that plays a vital role in the regulation of muscle contraction. Fast skeletal muscles express 2 Tpm isoforms, α (Tpm 1.1) and ß (Tpm 2.2), resulting in the existence of 2 forms of dimeric Tpm molecule: αα-homodimer and αß-heterodimer. ßß-Homodimer is unstable and absent in the native state, despite which most of the studies of myopathy-relating Tpm mutations have been performed on the ßß-homodimer. Here, we applied different methods to investigate the effects of myopathic mutations R133W and N202K in the ß-chain of Tpm on properties of αß-heterodimers and to compare them with the features of ßß-homodimers with the same mutations. The results show that properties of αß-Tpm and ßß-Tpm with substitutions in the ß-chain differ significantly, and this indicates that the effects of myopathic mutations in the Tpm ß-chain should be studied on the Tpm αß-heterodimer.-Bershitsky, S. Y., Logvinova, D. S., Shchepkin, D. V., Kopylova, G. V., Matyushenko, A. M. Myopathic mutations in the ß-chain of tropomyosin differently affect the structural and functional properties of ßß- and αß-dimers.

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle.

The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeFx complex (stable analog of S1∗-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeFx complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1.

Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.