Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity

Abstract

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys³²⁶ and Lys³²⁸, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca²⁺ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys³²⁶ and Lys³²⁸ are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca²⁺ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys³²⁶ and Lys³²⁸, also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca²⁺-dependent, <i>in vitro</i> motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca²⁺ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys³²⁸, modulates muscle contraction via disrupting inhibitory Tpm positioning.