The reason for the low Ca 2+ -sensitivity of thin filaments associated with the Glu41Lys mutation in the TPM2 gene is “freezing” of tropomyosin near the outer domain of actin and inhibition of actin monomer switching off during the ATPase cycle

The Primary Causes of Muscle Dysfunction Associated with the Point Mutations in Tpm3.12; Conformational Analysis of Mutant Proteins as a Tool for Classification of Myopathies

“…Specifically, E139del, Q147P and E41K mutations lead to a decrease in the number of strong-binding myosin heads in the ATPase cycle, while E173A and R90P mutations cause an increase in the number of such heads (Figure 6 and Figure 8). The E117K mutation associated with NM [70] appears to bring about a stronger reduction in the number of myosin heads strongly associated with actin at high Ca 2+ [74] than has been found for E139del [5], Q147P [68], and E41K [73] mutations. Specific changes in the behavior of tropomyosin, actin and myosin have been also revealed in the presence of the R91G mutation [67], which leads to the appearance of distal arthrogryposis (DA) [69].…”

“…Conversely, the mutations can decrease the relative number of myosin heads strongly bound to F-actin. It was found that at the AM state, the amount of the myosin heads strongly bound to actin decreased for the thin filaments containing E41K [73], or E117K mutations in β-Tpm [74], or R167H mutation in α-Tpm [54] (Table 1). This reduction was correlated with a decrease in the Ca 2+ -sensitivity, shown for the E41K and E117K mutations [9].…”

Anin vivoapproach to characterize novel variants associated with musculoskeletal disorders

“…Thin filament motility assays uncovered the biochemical properties of TPM2 variants in response to Ca 2+ , and the pathogenic variants tested so far have shown increased as well as reduced Ca 2+ sensitivity (Table 1). The basis for Ca 2+ sensitivity is thought to reside in the flexibility or rigidity of the TPM2 dimer, which correlates with the ability of troponin and myosin to shift tropomyosin away from actin (Avrova et al, 2018;Borovikov et al, 2020;Borovikov et al, 2017a;Borovikov et al, 2017b;Karpicheva et al, 2020).…”

“…Actin motility assays have shown that some TPM2 variants increase Ca 2+ sensitivity, causing maximum actin motility to be reached at comparatively low Ca 2+ concentrations, while other variants reduce Ca 2+ sensitivity (Table 1) (Borovikov et al, 2015;Marston et al, 2013;Marttila et al, 2012;Ochala et al, 2010). The addition of fluorescent probes and proteins to actin motility assays revealed that the Ca 2+ sensitivity of TPM2 variants correlates with the ability of troponin and myosin to shift tropomyosin away from actin, and that pathogenic substitutions alter tropomyosin flexibility (Avrova et al, 2018;Borovikov et al, 2020;Borovikov et al, 2017a;Borovikov et al, 2017b;Karpicheva et al, 2020). Since tropomyosin often exists as a heterodimer, TPM2 variants likely act as gain-of-function mutations by altering Ca 2+ sensitivity when dimerized with wild-type isoforms (Avrova et al, 2018;Borovikov et al, 2020;Matyushenko et al, 2019).…”

“…Extensive biochemical studies have been used to understand the gain-of-function phenotypes induced by TPM2 variants. Thin filaments, or even entire muscle fibers, can be reconstituted in vitro to assay myosin driven actin motility (Avrova et al, 2018;Borovikov et al, 2020;Borovikov et al, 2017a;Borovikov et al, 2017b;Karpicheva et al, 2020;Marston et al, 2013;Ochala et al, 2010). Reconstituted thin filaments contain actin, tropomyosin, and troponin, such that actin motility can be measured in response to a Ca 2+ gradient.…”