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

Familial hypertrophic cardiomyopathy (FHC) is a genetically transmitted disease that causes progressive remodeling of the myocardium and may result in sudden death. FHC is caused by mutations in sarcomeric proteins including the thin filament regulatory proteins troponin (Tn) and tropomyosin (Tm) (1-3). The single site mutations, D175N and E180G, in cardiac α-tropomyosin have been the subject of several early studies (4, 5), including force measurements on fibers (6, 7), in vitro motility assays (8), ATPase measurements on Tm mutants exchanged into myofibrils (9), physiological measurements on mouse model hearts (10) and myosin subfragment 1 (S1) binding kinetics using reconstituted thin filaments (11). These studies suggest that incomplete relaxation of the heart at the low Ca2+ concentrations present during diastole coupled with the increased Ca2+-sensitivity can cause some of the phenotypes observed in the disease. In most cases the Ca+2 -sensitivity increase was greater for E180G than D175N. However, the molecular basis for the in vivo abnormal functional properties of these Tm mutants is not known. Our early study on the properties of the D175N and E180G Tm mutants showed that: 1) the binding of these mutants to actin in the absence and presence of Tn is weakened (12), (note label mixup, (13)); 2) the thermal stability of E180G is decreased; 3) the region of the molecule near position 190 is destabilized. The greatest effects occur for the E180G FHC mutant. More recent biochemical studies have verified the weakened binding to actin (14). These mutations are located outside of the hydrophobic region of the coiled-coil but may be involved in ionic interactions across the chains (15). Thus, the single site mutations D175N and E180G dramatically affect the global properties of Tm, but the underlying mechanisms are not clear. It is also unknown how these effects cause the FHC phenotype. Tm is a coiled-coil molecule stabilized primarily by hydrophobic side-chain interactions between the two parallel α-helical polypeptide chains. In the actinTmTn thin filament, Tm acts in two ways: 1) by sterically inhibiting the interaction of myosin heads with actin at low Ca2+ and 2) by inhibiting the transition of myosin heads on actin to the force-generating state. This is the basis of the 3-state model consisting of Blocked (− Ca2+), Closed (Ca+2-indiuced) and Open (myosin-induced) (16) and is correlated with 3 positions of Tm on actin (17). The 3-state model has provided a useful framework for understanding normal muscle regulation. Recently, we proposed the inclusion of a 4th state, an active state present in the absence of Ca2+ produced by strong-binding myosin heads (18) in order to help understand how mutations, involving most of the sarcomeric muscle proteins, can cause cardiac diseases such as familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM). The FHC effects are mimicked by NEM-S1 (19), a strong binding analogue of S1 which does not dissociate from actin in the presence of ATP, indicating that the increase in Ca2+-sensitivity involves myosin. In this work, we showed that substitution of Gly and Gln neutral amino acids for negatively charged Glu and Asp, respectively, on the outside of the Tm coiled coil affects the protein local stability about 50 residues away, a phenomenon of long range information transfer. The decreased stability of the FHC mutants suggests a greater flexibility of Tm on the thin filament which may be important in their malfunction. The results are discussed within the framework of the 4-state model recently published whereby the contribution of an active state in the absence of Ca2+ can explain several effects produced by strong binding myosin heads (18).