Biochemical and Biophysical Research Communications
The molecular mechanism of muscle dysfunction associated with the R133W mutation in Tpm2.2
Graphical abstract
Introduction
It is believed that the interactions of Tpm, troponin (TN), and actin regulate muscle contraction in response to Ca2+ [1]. The electrostatic nature of the actin-Tpm interaction and flexibility of actin and Tpm [2] can explain the dynamic displacement of Tpm relative to the outer and inner domains of actin (between the blocked, closed and open positions) on the actin surface during contraction [3]. The change in the position of the Tpm relative to the inner domain of actin is due to the difference between tropomyosin and F-actin in their bending flexibility (therefore, in variation in the persistent lengths of these proteins [3,4]), which presumably cause azimuthal shift of the Tpm strands [[3], [4], [5]]. At low Ca2+, TN-I interacts with actin, switching thin filaments off [5], which leads to spatial rearrangement and an increase in the persistence length of the actin filament [4]. At the same time, the persistence length of the Tpm decreases [4] and limits the Tpm in a position close to the outer domain of actin to the “blocked” position [5]. In this state of the thin filament (the “OFF” state) [5], the strong connection of the myosin with actin is inhibited [1]. When Ca2+ binds to TN-C, some actin monomers change their conformation to the switched-on [6], and the persistence length of the actin filament decreases [4]. The persistence length of the Tpm increases, and Tpm moves towards the inner domain of actin [4,6], exposing a part of the myosin-binding site (“closed” position) [5]. When the myosin heads are strongly bound to the F-actin filament, the actin monomers are switched-on [6], the persistence length of the actin filament decreases, and that of Tpm increases [4]. In this state (the “ON” state), the Tpm strand completely exposes the binding sites of myosin to F-actin and, therefore, initiates muscle contraction [5].
In skeletal muscles there are three main tropomyosin isoforms, α, β and γ, which are encoded by the TPM1, TPM2 and TPM3 genes, respectively [7]. Tropomyosin exists as a parallel a-helical coiled-coil dimer and each of the three Tpm isoforms can form either homo- or heterodimers. Mutations in the Tpm genes give rise to a wide spectrum of clinically, histologically and genetically variable neuromuscular or cardiovascular disorders [[8], [9], [10]]. TPM2 mutations have been shown to cause distal arthrogryposis (characterized by distal limb deformities) [11,12], nemaline myopathy (characterized by nemaline bodies on muscle biopsy), congenital fibre-type disproportion and cap disease (characterized by cap-like structures located under the sarcolemma) [8]. Functional analyses have suggested that certain mutations cause a hypercontractile phenotype that is characterized by a higher Ca2+-sensitivity of myofilaments and a slightly higher maximum speed of their sliding in motility assay [9]. Conversely, other Tpm mutations demonstrate hypocontractile phenotype in which there is lower myofilament Ca2+-sensitivity, reduced sliding speeds in motility assay and reduced cross-bridge cycling rate [9]. An example of a mutation of the latter type is TPM2 R133W that causes nemaline myopathy or congenital fibre-type disproportion [8]. The molecular mechanisms underlying the specific dysfunction remain obscure.
The goal of the present study was to investigate the effect of the R133W mutation present in αβ- or ββ-Tpm on Ca2+-dependent changes in the binding of myosin heads to actin, the position of the mutant Tpms and the switching on of actin monomers during the ATPase cycle. It was observed that at high and low Ca2+ the R133W mutation causes a shift of αβ- or ββ-Tpm towards the outer edge of actin monomers, which inhibits the strong binding of the myosin heads to actin and the switching of actin monomers on during the ATPase cycle. This may represent the molecular mechanism underlying the decreased Ca2+-sensitivity and muscle weakness that are typical for nemaline myopathy [4,8].
Section snippets
Using of experimental animals
A source of biological material for this work was New Zealand white male rabbits. The animals were cared for and sacrificed in accordance with the protocols approved by the Ethics Committee for Animal Care and Usage of the Institute of Cytology of the Russian Academy of Science (Assurance Identification Number F18-00380, valid until October 31, 2022).
Preparation of proteins and their labeling by fluorescent probes
Myosin and TN were isolated from fast skeletal muscles by using standard procedures [11,12]. Treatment of myosin with α-chymotrypsin for 20 min at
Ghost muscle fibres reconstituted using 1,5-IAEDANS-labeled myosin heads as a model for investigation of contractile dysfunction
In this work we have reconstructed the thin filament in ghost muscle fibres using endogenous F-actin and exogenous Tpm and TN, decorated the filaments with S1 (Supplementary Fig. A) and mimicked several steps of ATP hydrolysis (see Material and methods). S1 was labeled with the fluorescent probe 1,5-IAEDANS that allowed detection of the changes in spatial arrangement and mobility of the myosin heads and the bending flexibility (or elastic modulus [19]) of F-actin in the muscle fibres [4]. S1
References (21)
- et al.
The relationship between curvature, flexibility and persistence length in the tropomyosin coiled-coil
J. Struct. Biol.
(2010) Switching muscles on and off in steps: the McKillop-Geeves three-state model of muscle regulation
Biophys. J.
(2017)- et al.
Modulation of the effects of tropomyosin on actin and myosin conformational changes by troponin and Ca2+
Biochim. Biophys. Acta
(2009) - et al.
Preparation of myosin and its subfragments from rabbit skeletal muscle
Methods Enzymol.
(1982) Preparation of TN and its subunits
Methods Enzymol.
(1982)- et al.
Polarization of fluorescence from single skinned glycerinated rabbit psoas fibres in rigor and relaxation
Biochim. Biophys. Acta
(1977) - et al.
Fluorescence depolarization of actin filaments in reconstructed myofibres: the effect of S1 or pPDM-S1 on movements of distinct areas of actin
Biophys. J.
(2004) - et al.
Conformational changes of F-actin in myosin-free ghost single fibre induced by either phosphorylated or dephosphorylated heavy meromyosin
Biochim. Biophys. Acta
(1987) - et al.
Polarized fluorescence from ε-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin
J. Mol. Biol.
(1978) - et al.
Regulation of contraction in striated muscle
Physiol. Rev.
(2000)
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