Mohammed EL-Mezgueldi

The effect of mutations in alpha tropomyosin (E40K and E54K), that cause familial dilated cardiomyopathy, on the regulatory mechanism of cardiac muscle thin filaments

E40K and E54K mutations in α-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser α-tropomyosin (AS-α-Tm) expressed in Escherichia coli, that both mutations decrease Ca2+ sensitivity. E40K also reduces Vmax of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-α-Tm, and the two dilated cardiomyopathy-causing mutants. AS-α-Tm has a lower cooperative unit size (6.5) than native α-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the KT value was the same for all actin-Tm species; however, the KT value of actin-Tm-troponin at pCa 5 was 50% less for AS-α-Tm E40K than for AS-α-Tm and AS-α-Tm E54K. Kb, the “closed” to “blocked” equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-α-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-α-Tm E54K, we observed a unique new domain at 55 °C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.

The Effects of Smooth Muscle Calponin on the Strong and Weak Myosin Binding Sites of F-actin

We have investigated the mechanism of inhibition of the actomyosin MgATPase by the smooth muscle protein calponin. We have shown previously the specific interaction of calponin with Glu334 of actin (EL-Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). This residue is within the sequence 332-334, which has been proposed to be an important part of the strong myosin binding site (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65). Therefore, we suggested that calponin will affect the strong binding actin-myosin interaction. To test this hypothesis we have investigated the effect of calponin on the strong binding of S-1·MgAMP-PNP (5′-adenylyl imidodiphosphate) and on the weak binding of S-1·MgADP·Pi to actin. We found that an inhibitory concentration of calponin decreased the binding of S-1·MgAMP-PNP to actin but had no effect on the binding of S-1·MgADP·Pi. Similar results were obtained with skeletal muscle and smooth muscle S-1. In competition experiments calponin was found to displace S-1·MgAMP-PNP and S-1·MgADP but not S-1·MgADP·Pi from the actin filament. S-1 displaced calponin from actin in the rigor state, in the presence of MgADP, and in the presence of MgAMP-PNP. We conclude that calponin inhibits the actin activated S-1 ATPase by blocking a strong S-1 binding site on actin and does not block the weak binding site.

The Mechanism of Smooth Muscle Caldesmon-Tropomyosin Inhibition of the Elementary Steps of the Actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1·ADP; however the rate of phosphate release from the actin-S1·ADP·Pi complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.

Role of Caldesmon in the Ca2+ Regulation of Smooth Muscle Thin Filaments EVIDENCE FOR A COOPERATIVE SWITCHING MECHANISM

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle αα-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca2+-calmodulin-caldesmon activation of actin-tropomyosin.

Cooperative inhibition of actin filaments in the absence of tropomyosin.

We measured the inhibition of actin activated myosin subfragment-1 MgATPase activity in a solution containing no added KCl (5 mM PIPES.K2 (pH 7.1), 2.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, 5 mM MgATP). Maximal inhibition was observed with substoichiometric concentrations of caldesmon, caldesmon domain 4, troponin and troponin I. In six experiments using different preparations of actin, S-1 and caldesmon 50% inhibition required 0.09 ± 0.01 (sem) caldesmon added per actin. This compares with 0.66 ± 0.32 (sem, n = 5) caldesmon per actin for 50% inhibition in the presence of 60 mM KCl. With caldesmon domain 4, 50% inhibition was achieved with 0.17 ± 0.08 (n = 11) domain 4 added per actin. We measured the amount of caldesmon bound at the same time as inhibition. Complete inhibition of actin activated ATPase needed only one caldesmon bound per 5.0 ± 0.5 (sem, n = 5) actin monomers or one caldesmon domain 4 bound per 3.9 ± 0.6 (sem, n = 3) actin monomers at zero KCl. We conclude that under these conditions inhibition of actin is cooperative despite the absence of tropomyosin. We measured the effect of caldesmon inhibition upon S-1 binding to actin. S-1.ADP.Pi (weak binding) was not affected by caldesmon concentrations giving 80% inhibition, however S-1.ADP (strong binding) was highly cooperative, being very weak at <0.3 μM but indistinguishable from uninhibited actin at >2 μM S-1.ADP. We conclude that actin can exist in two activity states corresponding to the ‘on’ and ‘off’ states of actin–tropomyosin and inhibitory proteins function as allosteric-cooperative inhibitors of actin. The implications of these findings for the role of tropomyosin in thin filament regulation are discussed.

Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction

We have investigated the functional properties of a mutant (Cg1) derived from the C‐terminal 99 amino acids of chicken caldesmon, 658–756 (658C) where the sequence 691glu‐trp‐leu‐thr‐lys‐thr696 is changed to pro‐gly‐his‐tyr‐asn‐asn. Cg1 bound Ca2+‐calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+‐calmodulin with the Trp‐722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+‐calmodulin is not able to reverse the Cg1‐induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin‐tropomyosin activation of myosin ATPase. The results are interpreted in terms of multi‐site attachment of actin and Ca2+‐calmodulin to overlapping sequences in caldesmon domain 4b.