The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

Molluscan twitchin can control actin-myosin interaction during ATPase cycle

The effect of twitchin, a thick filament protein of molluscan muscles, on actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using fluorescent probes specifically bound to Cys707 of myosin subfragment-1 and Cys374 of actin incorporated into ghost muscle fibers. The multi-step changes in mobility and spatial arrangement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle have been revealed. For the first time, the inhibition of movement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle and the decrease in the myosin head and actin affinity in the presence of unphosphorylated twitchin have been demonstrated. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reversed this effect. These data imply a novel property of twitchin consisting in its ability to regulate in a phosphorylation-dependent manner the actin-myosin interaction during the ATPase cycle by the inhibition of transformation of the weak-binding actomyosin states into the strong-binding ones.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift tropomyosin strands further towards the open position and to change the affinity of tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca²⁺-sensitivity caused by these mutations which initiates the disease remodeling.     

A new property of twitchin to restrict the “rolling” of mussel tropomyosin and decrease its affinity for actin during the actomyosin ATPase cycle

A new evidence on the regulatory function of twitchin, a titin-like protein of molluscan muscles, at muscle contraction has been obtained at studying the movements of IAF-labeled mussel tropomyosin in skeletal ghost fibers during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs. For the first time, myosin-induced multistep changes in mobility and in the position of mussel tropomyosin strands on the surface of the thin filament during the ATP hydrolysis cycle have been demonstrated directly. Unphosphorylated twitchin shifts the tropomyosin towards the position typical for muscle relaxation, decreases the tropomyosin affinity to actin and inhibits its movements during the ATPase cycle. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reverses this effect. These data imply that twitchin is a thin filament regulator that controls actin-myosin interaction by “freezing” tropomyosin in the blocked position, resulting in the inhibition of the transformation of weak-binding states into strong-binding ones during ATPase cycle.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

Yeast calmodulin known to be ubiquitylated in vivo in a Ca²⁺ dependent manner has long remained an orphan substrate. Here we identify Saccharomyces cerevisiae Asr1p as an ubiquitin E3 ligase for yeast calmodulin, a protein involved in calcium signaling. A short region within Asr1p-C harboring two putative calmodulin-binding motifs is sufficient and necessary for interaction with calmodulin. The interaction is direct, occurs in vivo and depends on physiological concentrations of Ca²⁺. A minimal set of purified proteins including Asr1p E3 ligase was sufficient for in vitro ubiquitylation of calmodulin, a reaction that required a functional Asr1p Ring domain. We propose a role of the Asr1p E3 ligase activity in coping with stress.     

Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.     

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM^∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.     

Caldesmon inhibits the actin-myosin interaction by changing its spatial orientation and mobility during the ATPase activity cycle

Orientation and mobility of acrylodan fluorescent probe specifically bound to caldesmon Cys580 incorporated into muscle ghost fibers decorated with myosin S1 and containing tropomyosin was studied in the presence or absence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. Modeling of various intermediate states of actomyosin has shown discrete changes in orientation and mobility of the dye dipoles which is the evidence for multistep changes in the structural changes of caldesmon during the ATPase hydrolysis cycle. It is suggested that S1 interaction with actin results in nucleotide-dependent displacement of the C-terminal part of caldesmon molecule and changes in its mobility. Thus inhibition of the actomyosin ATPase activity may be due to changes in caldesmon position on the thin filament and its interaction with actin. Our new findings described in the present paper as well as those published recently elsewhere might conciliate the two existing models of molecular mechanism of inhibition of the actomyosin ATPase by caldesmon.     

Autonomic cardiac control in animal models of cardiovascular diseases II. Variability analysis in transgenic rats with alpha-tropomyosin mutations Asp175Asn and Glu180Gly.

Animal models of cardiovascular diseases allow to investigate relevant pathogenetic mechanisms in detail. In the present study, the mutations Asp175Asn and Glu180Gly in alpha-tropomyosin (TPM1), known cause familiar hypertrophic cardiomyopathy (FHC) were studied for changes in hemodynamic parameters and spontaneous baroreflex regulation in transgenic rats in comparison to transgenic and non-transgenic controls by telemetry. Heart rate variability (HRV) and blood pressure variability (BPV) were analyzed using time- and frequency domain, as well as non-linear measures. The dual sequence method was used for the estimation of the baroreflex regulation. In transgenic rats harboring mutated TPM1, changes in HRV were detected during exercise, but not at rest. Both mutations, Asp175Asn and Glu180Gly, caused increased low frequency power. In addition, in animals with mutation Asp175Asn a reduced total HRV was observed. BPV did not show any differences between all transgenic animal lines. During exercise, a strong increase in the number of bradycardic and tachycardic fluctuations accompanied with decreased baroreflex sensitivity (BRS) was detected in animals with either TPM1 mutation, Asp175Asn or Glu180Gly. These data suggest, that the analysis of cardiac autonomic control, particularly of baroreflex regulation, represents a powerful non-invasive approach to investigate the effects of subtle changes in sarcomeric architecture on cardiac physiology in vivo. In case of mutations Asp175Asn or Glu180Gly in TPM1, early detection of alterations in autonomic cardiac control could help to prevent sudden cardiac death in affected persons.     

The interaction between the mitochondrial ATPase (F1) and the ATPase inhibitor

1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F₁) non-competitively.2. The interaction between inhibitor and inhibitor-depleted F₁ or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation.3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP.4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F₁. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler.5. Evidence is presented for the existence of a non-inhibited intermediate F₁-inhibitor complex.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

The NMR solution structure of subunit G (G61-101) of the eukaryotic V1VO ATPase from Saccharomyces cerevisiae

Subunit G is an essential stalk subunit of the eukaryotic proton pump V₁V_O ATPase. Previously the structure of the N-terminal region, G_1-59, of the 13 kDa subunit G was solved at higher resolution. Here solution NMR was performed to determine the structure of the recombinant C-terminal region (G_61-101) of subunit G of the Saccharomyces cerevisiae V₁V_O ATPase. The protein forms an extended α-helix between residues 64 and 100, whereby the first five- and the last residues of G_61-101 are flexible. The surface charge distribution of G_61-101 reveals an amphiphilic character at the C-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The hydrophobic surface pattern is mainly formed by alanine residues. The alanine residues 72, 74 and 81 were exchanged by a single cysteine in the entire subunit G. Cysteines at positions 72 and 81 showed disulfide formation. In contrast, no crosslink could be formed for the mutant Ala74Cys. Together with the recently determined NMR solution structure of G_1-59, the presented solution structure of G_61-101 enabled us to present a first structural model of the entire subunit G of the S. cerevisiae V₁V_O ATPase.     

The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F₁) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions.2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities.3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP.4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation.5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor.6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles.7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F₁-aurovertin complex.8. It is suggested that the ATPase inhibitor brings F₁ in a conformation denoted 1F₁ that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.     

The cell cycle of epidermal cells during reprogramming in the pupa of Galleria

The epidermal cell cycle of the pupal mesonotum of Galleria was investigated by the determination of mitotic indices, [³H]thymidine incorporation and flow-cytophotometric analysis during the first 48 h after pupation.Immediately after the pupal ecdysis nearly all epidermal cells are arrested in G2. Thereafter only a few mitoses occur, leading to a slow increase in the number of G1 nuclei. With the onset of a mitotic wave at a pupal age of 21 h this increase becomes more rapid. On day 2, the cell population reaches a plateau in the number of G1 (resp. G2) cells, reflecting a steady state between mitotic activity and DNA synthesis.A comparison of these cell cycle changes with known data of the time course of reprogramming and ecdysteroid titre leads to the conclusion that there is no causal relationship between DNA synthesis and cellular determination in the sense of a quantal cell cycle, and that DNA synthesis can precede the definite rise in ecdysteroid titre.     

Interaction between subunit C (Vma5p) of the yeast vacuolar ATPase and the stalk of the C-depleted V1 ATPase from Manduca sexta midgut

Projection maps of a V₁-Vma5p hybrid complex, composed of subunit C (Vma5p) of Saccharomyces cerevisiae V-ATPase and the C-depleted V₁ from Manduca sexta, were determined from single particle electron microscopy. V₁-Vma5p consists of a headpiece and an elongated wedgelike stalk with a 2.1×3.0 nm protuberance and a 9.5×7.5 globular domain, interpreted to include Vma5p. The interaction face of Vma5p in V₁ was explored by chemical modification experiments.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Caldesmon inhibits the rotation of smooth actin subdomain-1 and alters its mobility during the ATP hydrolysis cycle

Smooth muscle thin filaments have been reconstituted in muscle ghost fibers by incorporation of smooth muscle actin, tropomyosin and caldesmon. For the first time, rotation of subdomain-1 and changes of its mobility in IAEDANS-labeled actin during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs have been demonstrated directly. Binding of caldesmon altered the mobility and inhibited the rotation of actin subdomain-1 during the transition from AM∗∗·ADP·Pi to AM state, resulting in inhibition of both strong and weak-binding intermediate states. These new results imply that regulation of actomyosin interaction by caldesmon during the ATPase cycle is fulfilled via the inhibition of actin subdomain-1 rotation toward the periphery of the thin filament, which decreases the area of the specific binding between actin and myosin molecules and is likely to underlie at least in part the mechanism of caldesmon-induced contractility suppression.     

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1-59) of the Saccharomyces cerevisiae V1VO ATPase

Understanding the structural traits of subunit G is essential, as it is needed for V₁V_O assembly and function. Here solution NMR of the recombinant N- (G_1-59) and C-terminal segment (G_61-114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G_1-59 only. The residues of G_1-59 involved in d binding are Gly7 to Lys34. The structure of G_1-59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G_1-59 are flexible. The surface charge distribution of G_1-59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G_1-59-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E_18-38 of subunit E of yeast V-ATPase and the presently solved structure of G_1-59, both proteins have been docked and binding epitopes have been analyzed.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.