Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.     

Identification of a Conserved Calmodulin-Binding Motif &;#x220A; the Sequence of F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase Inhibitor Protein

The natural inhibitor proteins IF₁ regulate mitochondrial F₀F₁ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF₁, two bovine IF₁ synthetic peptides, as well as two homologous proteins from yeast, namely IF₁ and STF₁. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF₁ interaction is Ca²⁺-dependent. Bovine and yeast IF₁ have intermediate binding affinity for CaM, while the K_d (dissociation constant) of the STF₁-CaM interaction is slightly higher. Binding studies of CaM with bovine IF₁ synthetic peptides allowed us to identify bovine IF₁ sequence 33–42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF₁ and STF₁ sequences. In addition, the same region in bovine IF₁ has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF₁ but not in STF₁. Based on the pH and Ca²⁺ dependence of IF₁ interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF₁ trafficking or additional IF₁ roles not yet clarified.     

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F₁F₀-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF₁) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF₁ protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F₁F₀ mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F₁ portion. In addition, F₁F₀ dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.     

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α_DP- and β_DP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β_E-subunits. The nucleotide binding site in β_E-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.     

Effects of Zn2+ on the activity and binding of the mitochondrial ATPase inhibitor protein,IF1

Zn²⁺ caused a noninhibitory binding of IF₁ to mitochondrial membranes in both rabbit heart SMP and intact rabbit heart mitochondria. This Zn²⁺-induced IF₁ binding required the presence of at least trace amounts of MgATP and was essentially independent of pH between 6.2 and 8.2. Addition of Zn²⁺ after the formation of fully inhibited IF₁-ATPase complexes very slowly reversed IF₁-mediated ATPase inhibition without causing significant IF₁ release from the membranes. When Zn²⁺ was added during the state 4 energization of ischemic mitochondria in which IF₁ was already functionally bound, it slowed somewhat energy-driven ATPase activation. This slowing was probably due to the fairly large depressing effect Zn²⁺ had upon membrane potential development, but Zn²⁺ did not decrease the degree of ATPase activation eventually reached at 20 min of state 4 incubation. Zn²⁺ also preempted normal IF₁ release from the membranes, causing what little inhibitor that was released to rebind to the enzyme in noninhibitory IF₁-ATPase complexes. The data suggest that IF₁ can interact with the ATPase in two ways or through two kinds of sites: (a) a noninhibitory interaction involving a noninhibitory IF₁ conformation and/or and IF₁ docking site on the enzyme and (b) an inhibitory interaction involving an inhibitory IF₁ conformation and/or a distinct ATPase activity regulatory site. Zn²⁺ appears to have the dual effect of stabilizing the noninhibitory IF₁-ATPase interaction and possibily a noninhibitory IF₁ conformation while concomitantly preventing the formation of an inhibitory IF₁-ATPase interaction and possibly an inhibitory IF₁ conformation, regardless of pH. While the data do not rule out direct effects of Zn²⁺ on either free IF₁ or the free enzyme, they suggest that Zn²⁺ cannot interact readily with either the inhibitor or the enzyme once functional IF₁-ATPase complexes are formed.     

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma,epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase

The location of the endogenous inhibitor protein ( IF₁) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F₁I and intact F₁F₀I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of –IF₁, IF₁–IF₁, –IF₁, and –IF₁ cross-linkages in soluble F₁I and in native F₁F₀I complexes. Cross-linking blocked the release of IF₁ from its inhibitory site and therefore the activation of F₁I and F₁F₀I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF₁ is at a distance 12 Å,to and subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF₁ inhibits conformational changes of the catalytic subunits, the inhibitory mechanism of IF₁ may involve the interference with rotation of the central stalk.     

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF₁, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O₂) unexpectedly showed that Δψ_m is preserved independently of the presence of IF₁ and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ_Η⁺ with the FCCP uncoupler, the IF₁-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF₁ overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF₁ overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.     

IF<Subscript>1</Subscript> distribution in HepG2 cells in relation to ecto–F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase and calmodulin

F₀F₁ATPsynthase is now known to be expressed as a plasma membrane receptor for several extracellular ligands. On hepatocytes, ecto–F₀F₁ATPsynthase binds apoA–I and triggers HDL endocytosis concomitant with ATP hydrolysis. Considering that inhibitor protein IF₁ was shown to regulate the hydrolytic activity of ecto–F₀F₁ATPsynthase and to interact with calmodulin (CaM) in vitro, we investigated the subcellular distributions of IF₁, calmodulin (CaM), OSCP and β subunits of F₀F₁ATPsynthase in HepG2 cells. Using immunofluorescence and Western blotting, we found that around 50% of total cellular IF₁ is localized outside mitochondria, a relevant amount of which is associated to the plasma membrane where we also found Ca²⁺–CaM, OSCP and β. Confocal microscopy showed that IF₁ colocalized with Ca²⁺–CaM on plasma membrane but not in mitochondria, suggesting that Ca²⁺–CaM may modulate the cell surface availability of IF₁ and thus its ability to inhibit ATP hydrolysis by ecto–F₀F₁ATPsynthase. These observations support a hypothesis that the IF₁–Ca²⁺–CaM complex, forming on plasma membrane, functions in the cellular regulation of HDL endocytosis by hepatocytes.     

The Torque of Rotary F-ATPase Can Unfold Subunit Gamma If Rotor and Stator Are Cross-Linked

During ATP hydrolysis by F₁-ATPase subunit γ rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (αβ)₃. If the penultimate residue at the α-helical C-terminal end of subunit γ is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F₁ persists. This observation has been tentatively interpreted by the unfolding of the α-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit γ, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single α-helical, C-terminal portion of subunit γ (from γ285 to γ276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal α-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular α-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme''s torque.     

Effect of the protonmotive force on ATP-linked processes and mobilization of the bound natural ATPase inhibitor in beef heart submitochondrial particles

In an attempt to determine whether the natural ATPase inhibitor (IF₁) plays a role in oxidative phosphorylation, the time course of ATP synthesis and ATP hydrolysis in inside-out submitochondrial particles from beef heart mitochondria either possessing IF₁ (Mg-ATP particles) or devoid of IF₁ (AS particles) was investigated and compared to movements of IF₁, as assessed by an isotopic assay. The responses of the above reactions to preincubation of the particles in aerobiosis with NADH or succinate were as follows: (1) The few seconds lag that preceded the steady-rate phase of ATP synthesis was shortened and even abolished both in Mg-ATP particles and AS particles. The rate of ATP synthesis in the steady state was independent of the length of the lag. (2) ATPase was slowly activated, maximal activation being obtained after a 50-min preincubation; there was no direct link between the development of the protonmotive force (maximal within 1 sec) and ATPase activation. (3) Bound IF₁ was slowly released; the release of bound IF₁ as a function of the preincubation period was parallel to the enhancement of ATPase activity; the maximal amount of IF₁ released was a small fraction of the total IF₁ bound to the particles (less than 20%). (4) The double reciprocal plots of the rates of ATP and ITP hydrolysis vs. substrate concentrations that were curvilinear in the absence of preincubation with a respiratory substrate became linear after aerobic preincubation with the substrate. The data conclusively show that only ATPase activity in submitochondrial particles is correlated with the release of IF₁, and that the total extent of IF₁ release induced by respiration is limited. On the other hand, the kinetics of ATPase in control and activated particles are consistent with the existence of two conformations of the membrane-bound F₁-ATPase, directed to ATP synthesis or ATP hydrolysis and distinguishable by their affinity for IF₁.     

Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66

Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln¹⁰⁰ to Ile⁷³² of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold; domain A contained the enzyme''s catalytic module, comprising a (β/α)₈-barrel; and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, −4 to −1, in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp³⁸⁵ side chain. These structures collectively indicated that Asp³⁸⁵ was the catalytic nucleophile and that Glu⁴⁵³ was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme''s catalytic machinery.     

Purification,characterization and crystallization of the F-ATPase from Paracoccus denitrificans

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart

A method has been developed to allow the level of F₀F₁ATP synthase capacity and the quantity of IF₁ bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF₁ content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF₁ antibodies.Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF₁ content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF₁. In addition, both in vivo and in vitro, 1.3-1.4 mol of IF₁ was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF₁ in the F₀ sector.     

Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire

The membrane‐associated serine hydrolase, monoacylglycerol lipase (MGL), is a well‐recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGL's membrane association upon its structure–function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge‐neutral phospholipid bilayer nanodiscs enhanced hMGL's kinetic properties [apparent maximum velocity (V_max) and substrate affinity (K_m)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL–nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid‐domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzyme's substrate‐binding channel. Covalent modification of membrane‐associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane‐associated topology of active and inhibited, covalently‐modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzyme's conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.     

Control of rotation of the F<Subscript>1</Subscript>F<Subscript>O</Subscript>-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF<Subscript>1</Subscript> proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF₁ subunits. These proteins regulate the CCW F₁F_O-ATPase activity by blocking γ subunit rotation at the α_DP/β_DP/γ subunit interface in the F₁ domain. Recently, we discovered a unique natural F₁-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF₁, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the α_DP/β_DP/γ interface of the F₁-ATPase. In order to exert inhibition, IF₁ and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the α_DP/β_DP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F₁F_O-ATPase rotation, without affecting the CW-F₁F_O-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF₁, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF₁ inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.     

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase

(1) The natural ATPase inhibitor (IF₁) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF₁ polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF₁. The higher the salt concentration, the lower the aggregation state. Aggregation of IF₁ was decreased at slightly acidic pH. It increased with the concentration of IF₁ as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF₁ in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF₁ (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547–2551) was shown to have a limited effectiveness as a trap for IF₁. The reason was that IF₁ released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF₁ which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF₁ preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336–1344). The particles recovered in the sediment were deprived of IF₁ and could therefore be used for preparation of F₁-ATPase. The advantage of this method is that both IF₁ and F₁-ATPase can be prepared from the same batch of mitochondria.     

The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3

We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K_i value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.     

Content and binding characteristics of the mitochondrial ATPase inhibitor,IF1 in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms

We determined the IF₁ contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF₁-mediated inhibition of the ATPase activity of IF₁-depleted rabbit heart mitochondrial particles (RHMP) by IF₁-containing extracts from these same eight species. The IF₁ binding experiments were run at both species-endogenous IF₁ levels and at an IF₁ level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF₁ or either speciesendogenous or normalized levels of pig heart IF₁ were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF₁-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF₁ were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF₁-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF₁ inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF₁ inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF₁, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF₁, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF₁. In contrast, rat and mouse heart mitochondria contain only low levels of IF₁ which exhibit binding characteristics similar to those of the pig and rabbit heart inhibitor. The guinea pig is the only mammal thus far examined that contains a loweraffinity form of IF₁. In the present study we also determined the IF₁ contents and IF₁-to-F₁ ATPase activity ratios of cardiac muscle, skeletal muscle, liver, and brain mitochondria of rabbit, pigeon, and rat, species representative of the three homeothermic regulatory classes.