Isolated noncatalytic and catalytic subunits of F1-ATPase exhibit similar, albeit not identical, energetic strategies for recognizing adenosine nucleotides.

The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic ß-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase ß subunit, Biochemistry 49 (2010) 5258-5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the µM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme.

Different contribution of conserved amino acids to the global properties of triosephosphate isomerases.

It is generally assumed that the amino acids that exist in all homologous enzymes correspond to residues that participate in catalysis, or that are essential for folding and stability. Although this holds for catalytic residues, the function of conserved noncatalytic residues is not clear. It is not known if such residues are of equal importance and have the same role in different homologous enzymes. In humans, the E104D mutation in triosephosphate isomerase (TIM) is the most frequent mutation in the autosomal diseases named "TPI deficiencies." We explored if the E104D mutation has the same impact in TIMs from four different organisms (Homo sapiens, Giardia lamblia, Trypanosoma cruzi, and T. brucei). The catalytic properties were not significantly affected by the mutation, but it affected the rate and extent of formation of active dimers from unfolded monomers differently. Scanning calorimetry experiments indicated that the mutation was in all cases destabilizing, but the mutation effect on rates of irreversible denaturation and transition-state energetics were drastically dependent on the TIM background. For instance, the E104D mutation produce changes in activation energy ranging from 430 kJ mol(-1) in HsTIM to -78 kJ mol(-1) in TcTIM. Thus, in TIM the role of a conserved noncatalytic residue is drastically dependent on its molecular background. Accordingly, it would seem that because each protein has a particular sequence, and a distinctive set of amino acid interactions, it should be regarded as a unique entity that has evolved for function and stability in the organisms to which it belongs.

New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition.

CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM). OBJECTIVE: Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative. METHODS: We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM. RESULTS AND CONCLUSION: At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.

Ordered-domain unfolding of thermophilic isolated ß subunit ATP synthase.

The flexibility of the ATP synthase's ß subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated ß subunit (Tß) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tß shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the ß-sheet residual structure at high temperature. We determined that part of the stability origin of Tß is due to a transversal hydrophobic array that crosses the ß-barrel formed at the N-terminal domain and the Rossman fold of the nucleotide-binding domain (NBD), while the helix bundle of the C-terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase.

Crosstalk between the subunits of the homodimeric enzyme triosephosphate isomerase.

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.

The importance of arginine codons AGA and AGG for the expression in E. coli of triosephosphate isomerase from seven different species.

Rare arginine codons AGA and AGG affect the heterologous expression of proteins in Eschericha coli. The tRNAs necessary for protein synthesis are scarce in E. coli strain BL21(DE3) pLysS and plentiful in strain BL21(DE3) CodonPlus -RIL. We evaluated in both bacterial strains the effect of these rare codons on the expression of triosephosphate isomerases from 7 different species, whose sequences had different dispositions of rare arginine codons. The ratio of expressed protein (CP/Bl21) correlated with the number of rare codons. Our study shows that the number, position and particularities of the combination of rare Arg codons in the natural non-optimized sequences of the triosephosphate isomerases influence the synthesis of heterologous proteins in E. coli and could have implications in the selection of better sequences for engineering enzymes for novel or manipulated metabolic pathways or for the expression levels of non enzymatic proteins..

The interfaces of oligomeric proteins as targets for drug design against enzymes from parasites.

Millions of people worldwide are infected by some kind of parasite and millions are in risk of contracting infection. In addition, it is now accepted that parasites are rapidly developing resistance to drugs that a few years ago were effective. This gloom picture underscores the urgent need to develop new drugs against parasitic diseases. Fortunately, the important technological advances that have been made in the past years will, in principle, facilitate the discovery of new and effective agents against parasitic diseases. In many of the approaches for drug design the basic premise is the identification of a macromolecule that is central to the life of the parasite. Because the life of all living organisms depends on multiple protein-protein interactions and the function of oligomeric proteins, it is worthwhile to explore if protein interfaces could be exploited for drug design. Here we review some of the work that has been done in this direction, and attempt to call attention to the richness of protein-protein interfaces for the design of agents that could lead to the development of drugs against parasitic diseases.

A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.

Structural basis of human triosephosphate isomerase deficiency: mutation E104D is related to alterations of a conserved water network at the dimer interface.

Human triosephosphate isomerase deficiency is a rare autosomal disease that causes premature death of homozygous individuals. The most frequent mutation that leads to this illness is in position 104, which involves a conservative change of a Glu for Asp. Despite the extensive work that has been carried out on the E104D mutant enzyme in hemolysates and whole cells, the molecular basis of this disease is poorly understood. Here, we show that the purified, recombinant mutant enzyme E104D, while exhibiting normal catalytic activity, shows impairments in the formation of active dimers and low thermostability and monomerizes under conditions in which the wild type retains its dimeric form. The crystal structure of the E104D mutant at 1.85 A resolution showed that its global structure was similar to that of the wild type; however, residue 104 is part of a conserved cluster of 10 residues, five from each subunit. An analysis of the available high resolution structures of TIM dimers revealed that this cluster forms a cavity that possesses an elaborate conserved network of buried water molecules that bridge the two subunits. In the E104D mutant, a disruption of contacts of the amino acid side chains in the conserved cluster leads to a perturbation of the water network in which the water-protein and water-water interactions that join the two monomers are significantly weakened and diminished. Thus, the disruption of this solvent system would stand as the underlying cause of the deficiency.

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein.

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500-650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.

The inhibitor protein of the F1F0-ATP synthase is associated to the external surface of endothelial cells.

The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.

Structural energetics of MgADP binding to the isolated beta subunit of F1-ATPase from thermophilic Bacillus PS3.

The energetics of binding of MgADP to the isolated beta subunit of F(1)-ATPase from thermophilic Bacillus (Tbeta) was characterized by high-precision isothermal titration calorimetry. The reaction was enthalpically driven, with a DeltaCp of -36cal(molK)(-1). To gain insight into the molecular basis of this small DeltaCp, we analyzed the changes in accessible surface areas (DeltaASA) between the structures of empty and MgADP-filled beta subunits, extracted from the crystal structure of bovine heart F(1). Consistent with the experimental DeltaCp, the DeltaASA was small (-775A(2)). We used a reported surface area model developed for protein reactions to calculate DeltaCp and DeltaH from DeltaASA, obtaining good agreement with the experimental values. Conversely, using the same model, a DeltaASA of -770A(2) was estimated from experimental DeltaCp and DeltaH for the Tbeta-MgADP complex. Our structural-energetic study indicates that on MgADP binding the isolated Tbeta subunit exhibits intrinsic structural changes similar to those observed in F(1).

Interconversion between dimers and monomers of endogenous mitochondrial F1-inhibitor protein complexes and the release of the inhibitor protein. Spectroscopic characteristics of the complexes.

The F1-inhibitor protein complex (F1-IP) was purified from heart submitochondrial particles. Size exclusion chromatography of the endogenous complex showed that it contains dimers (D) and monomers (M) of F1-IP. Further chromatographic analysis showed that D and M interconvert. At high protein concentrations, the interconversion reaction is shifted toward the D species. The release of the inhibiting action of IP is faster at low than at high protein concentrations. During activation of F1, the M species accumulates through a process that is faster than the release of IP from F1. These findings indicate that the activation of F1-IP involves the transformation of D into M, which subsequently loses IP. The spectroscopic characteristics of D, M, and free F1 show that the binding of IP and dimerization modifies the fluorescence intensity of tyrosine residues and that of the single tryptophan of F1 which is far from the IP binding site.

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria.

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.