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- Monomeric versus decameric vanadate in the elucidation of muscle contraction regulation: a kinetic, spectroscopic and structural overviewPublication . Tiago, Teresa; Gutiérrez-Merino, Carlos; Aureliano, M.Vanadium (V) was rediscovered for biology as a “muscle inhibitor factor” when it was found in commercial ATP prepared from equine muscle almost thirty years ago. Since then it has been used as a molecular probe of the mechanisms of several enzyme reactions involving hydrolysis of phosphate ester bonds. Besides acting as a phosphate analogue, vanadate has also the potential to exhibit biological activities through oligomeric vanadate species. Among the vanadate oligomers, decavanadate is one of the most potent inhibitors and has revealed an excellent kinetic and spectroscopic probe. This is particularly relevant for myosin, the major muscle ATPase which along with actin is able to convert the chemical energy of ATP hydrolysis into mechanical work. Apparently, vanadate is able to populate different conformational states of the myosin ATPase cycle depending on its oligomerization state. While monomeric vanadate (VO4 3-) mimics the transition state for the g-phosphate hydrolysis blocking myosin in a pre-power stroke state, decameric vanadate (V10O28 6-) induces the formation of the intermediate myosin·MgATP·V10 complex blocking the actomyosin cycle in a pre-hydrolysis state. These recent findings, that are now reviewed, point out to the importance of taking into account vanadate species variety in studies describing the interaction of vanadate with biological systems and incite the use of decavanadate as a biochemical tool to the elucidation of muscle contraction regulation.
- Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activityPublication . Tiago, Teresa; Martel, Paulo; Gutiérrez-Merino, Carlos; Aureliano, M.Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V10O28 6−) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V10 binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V10 oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V10 to bind at the back-door, but only on the “open” structures where there is access to the phosphate binding-loop. It is suggested that V10 acts as a “back-door stop” blocking the closure of the 50- kDa cleft necessary to carry out ATP-γ-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V10 and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.
- Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and functionPublication . Tiago, Teresa; Palma, Pedro; Gutiérrez-Merino, Carlos; Aureliano, M.The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the effi ciency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modifi cation in the peroxynitrite-induced myosin functional impairment.
- Inhibition of skeletal muscle S1-myosin ATPase by peroxynitritePublication . Tiago, Teresa; S, Simão; Aureliano, M.; Martín-Romero, Francisco Javier; Gutiérrez-Merino, CarlosExposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg2+-ATPase activity, reaching 50% inhibition with 46.7 ( 8.3 íM SIN-1 for 8.7 íM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg2+-ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca2+-dependent and K+/EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg2+-ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys707 and Cys697), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5¢-dithiobis(2- nitrobenzoic acid) and by the parallel decrease of Cys707 labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg2+-ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADPâV1 to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.
- Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activityPublication . Tiago, Teresa; Aureliano, M.; Gutiérrez-Merino, CarlosDecameric vanadate (V10) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (Ki ) 0.27 ( 0.05 íM) in myosin subfragment-1 (S1). The binding of V10 to S1 can be monitored from titration with V10 of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N¢-(5-sulfo-1-naphthyl)- ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V10 binding site per monomer with a dissociation constant of 0.16-0.7 íM, indicating that S1 labeling with these dyes produced only a small distortion of the V10 binding site. The large quenching of AEDANSlabeled S1 fluorescence produced by V10 indicated that the V10 binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V10 to S1 is not competitive either with actin or with ADPâV1 or ADPâAlF4; (ii) the affinity of V10 for the complex S1/ADPâV1 and S1/ ADPâAlF4 is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 “back door” ligand P1P5-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V10 is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V10 to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.