Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

Abstract

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, Bfree^and Bblocked^, formed an asymmetric structure named the Binteracting-heads motif^(IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca 2+ -activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and posttetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

“…We used mant-ATP single turnover experiments to calculate the fraction of myosin heads hydrolyzing ATP at their normal basal (~0.03 s -1 ) rate versus their SRX (~0.003 s -1 ) rate. 24,41,42 A recent publication by Anderson et al 24 showed that the SRX corresponds to a folded-back (sequestered) state of myosin, possibly the IHM described previously 30,31 . Our studies show that the mutant 25-hep HMMs show a much greater fraction of myosin heads in the fast phase compared to WT 25-hep HMM, consistent with our hypothesis that these mutations disrupt the myosin intramolecular interactions that sequester heads in the SRX inactive state.…”

“…3a). If the myosin is in an open state, then its mant-nucleotide release rate in the absence of actin is higher (~0.03 s -1 ; 'fast') than that of the sequestered SRX (~0.003 s -1 ; 'slow') 30,41 . Fitting the mant-ATP fluorescence vs time data to a double exponential (equation 1) gives the fraction of myosin in the SRX slow phase.…”

“…While the high-resolution structure of such a sequestered form of human ß-cardiac myosin heads is yet to be determined, the best working structural hypothesis involves a known folded-back sequestered structure involving interactions between different subdomains of myosin referred to as the interacting heads motif (IHM; Fig. 1a) [29][30][31][32][33][34][35][36] . The IHM structure has an asymmetric conformation of the two S1 heads, one head thought to be blocked for actin interaction (the 'blocked head') and one which possibly could still interact with actin (the 'free head').…”

Direct Sarcomere Modulators Are Promising New Treatments for Cardiomyopathies

“…Dynamic and asymmetric interaction of the two heads in a pair of myosin II molecules forms a myosin interacting-heads motif (IHM) structure. During relaxation, there are two relaxation states of myosin dimers in striated muscles, including human cardiac muscle, depending on IHM interaction: the disordered relaxed (DRX) state and the ordered relaxed or super-relaxed (SRX) state [8,9] ( Figure 2). In the DRX state, the blocked head (BH) docked onto its S2 and actin-binding interface is positioned on the converter domain of the partner free head (FH).…”

“…The disordered relaxed (DRX) state and the ordered relaxed or super-relaxed (SRX) state [8,9] (Figure 2). In the DRX state, the blocked head (BH) docked onto its S2 and actin-binding interface is positioned on the converter domain of the partner free head (FH).…”