Long-range effects of familial hypertrophic cardiomyopathy mutations E180G and D175N on the properties of tropomyosin

Abstract

Cardiac α-tropomyosin (Tm) single-site mutations D175N and E180G cause familial hypertrophic cardiomyopathy (FHC). Previous studies have shown that these mutations increase both Ca(2+) sensitivity and residual contractile activity at low Ca(2+) concentrations, which causes incomplete relaxation during diastole resulting in hypertrophy and sarcomeric disarray. However, the molecular basis for the cause and the difference in the severity of the manifested phenotypes of disease are not known. In this work we have (1) used ATPase studies using reconstituted thin filaments in solution to show that these FHC mutants result in an increase in Ca(2+) sensitivity and an increased residual level of ATPase, (2) shown that both FHC mutants increase the rate of cleavage at R133, ~45 residues N-terminal to the mutations, when free and bound to actin, (3) shown that for Tm-E180G, the increase in the rate of cleavage is greater than that for D175N, and (4) shown that for E180G, cleavage also occurs at a new site 53 residues C-terminal to E180G, in parallel with cleavage at R133. The long-range decreases in dynamic stability due to these two single-site mutations suggest increases in flexibility that may weaken the ability of Tm to inhibit activity at low Ca(2+) concentrations for D175N and to a greater degree for E180G, which may contribute to differences in the severity of FHC.

Fig. 1

Effects of Tm-FHC mutants on actin-S1 ATPase activity. Data obtained by continuously monitoring Pi liberation (see Methods). A. Effects of Tm and Tn +/− Ca²⁺. ATPase was monitored during successive additions of S1, actin, Tm, Tn, EGTA to a solution containing, 0.1mM CaCl₂, 15 mM NaCl, 3 mM MgCl₂, 1 mM DTT and 1 mM ATP in buffer (10 mM HEPES, pH 7.5). Data were normalized to actin-S1 activity after subtracting the background S1 ATPase. B. ATPase vs. [Ca²⁺]. CaCl₂ was titrated into the reconstituted actinTmTn thin filament in buffer containing 1mM EGTA and 1 mM NTA. Data were normalized to the high [Ca²⁺] values fitted to the Hill equation. Background S1 alone ATPase was subtracted.

Fig. 1

Effects of Tm-FHC mutants on actin-S1 ATPase activity. Data obtained by continuously monitoring Pi liberation (see Methods). A. Effects of Tm and Tn +/− Ca²⁺. ATPase was monitored during successive additions of S1, actin, Tm, Tn, EGTA to a solution containing, 0.1mM CaCl₂, 15 mM NaCl, 3 mM MgCl₂, 1 mM DTT and 1 mM ATP in buffer (10 mM HEPES, pH 7.5). Data were normalized to actin-S1 activity after subtracting the background S1 ATPase. B. ATPase vs. [Ca²⁺]. CaCl₂ was titrated into the reconstituted actinTmTn thin filament in buffer containing 1mM EGTA and 1 mM NTA. Data were normalized to the high [Ca²⁺] values fitted to the Hill equation. Background S1 alone ATPase was subtracted.

Fig. 2

CD thermal unfolding profiles at 222 nm (Θ vs T) and derivatives (dΘ/dT) of theTm-FHC mutants and WT control. Samples contained 1 mg/mL protein in 100 mM NaCl, 10 mM HEPES buffer pH 7.5, 1mM DTT.

Fig. 3

Kinetics of trypsin digestion of FHC Tms and WT control. Top, 12.5% polyacrylamide SDS-gels quenched at indicated times; Bottom, densitometric analyses showing kinetics of change of gel bands. The densities were normalized to the density of Tm at 0 time.

Fig. 4

Kinetics of trypsin digestion of Tm-FHC mutants and WT control bound to actin. Left, 12.5% polyacriylamide SDS-gels quenched at indicated times: Right, densitometric analysis of kinetics of cleavage of Tm normalized to 0 time.

Fig. 5

Schematic of the course of cleavage by trypsin. Note the common pathway for all via initial cleavage at R133 and an additional pathway for E180G with cleavage at K233. Helical peptides are indicated as dimers. * indicates a “hot spot” where cleavage occurs preferentially.