Modulation of the Oligomerization State of the Bovine F1-ATPase Inhibitor Protein, IF1, by pH

Abstract

Bovine IF 1 , a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F 1 domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF 1 has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF 1 consisting of residues 44 -84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel ␣-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F 1 .The activity of the proton-pumping ATP synthase (F 1 F 0 -ATPase) found in mitochondria is regulated in vivo by ADP, a proton motive force (⌬H ϩ ) and by a natural protein inhibitor, IF 1 (1). Decrease of ⌬H ϩ can reverse the action of ATP synthase, making it hydrolyze ATP and pump protons out of the organelle. ATP hydrolysis is inhibited by the inhibitor protein, IF 1 , which binds to ATP synthase in a 1:1 stoichiometry (2). Binding depends on the presence of MgATP. Restoration of a proton motive force favoring ATP synthesis displaces IF 1 from its inhibitory site and releases bound ATP, indicating that IF 1 prevents product release (3, 4). The binding of IF 1 to ATP synthase is dependent on pH. Upon reduction of pH below neutrality, the inhibitory capacity of IF 1 increases (5). In respiring mitochondria, the pH is relatively low outside and relatively high inside the matrix where ATP is made. In the absence of oxygen or in the presence of an uncoupler of oxidative phosphorylation, ATP synthase is poised in the direction of ATP hydrolysis, and glycolysis becomes the only source of cellular ATP. The high rate of glycolysis results in reduction of cytosolic pH (6) which is transmitted to the mitochondrial matrix, promoting inhibition of ATP hydrolysis by IF 1 in order to preserve ATP.

Crystal structure of a conserved N-terminal domain of histone deacetylase 4 reveals functional insights into glutamine-rich domains

“…A single His-His packing interaction was previously observed in the Max homodimer (27). But in the present structure, a total of 20 histidine residues are buried in the HDAC4 four-helix bundle, suggesting that the assembly of the HDAC4 tetramer may be sensitive to pH changes (33). Indeed, we have observed that the HDAC4 tetramer dissociates into monomer and lower order oligomers when the pH is shifted below the pK a (pH 6.5) of histidine (data not shown).…”

Amyloid precursor protein and amyloid β-peptide bind to ATP synthase and regulate its activity at the surface of neural cells

“…IF 1 inhibits the activity of ATP synthase also at physiological pH 7.3, but to a lesser extent than at acidic pH. 46 Interestingly, the RHS amino acid sequence in the putative binding domain of Ab to ATP synthase subunit a shows a high degree of homology to the minimal inhibitory sequence of IF 1 . This sequence is also reported to be responsible for binding of Ab to a5b1 integrin.…”

An Inhibitor of the F1 Subunit of ATP Synthase (IF1) Modulates the Activity of Angiostatin on the Endothelial Cell Surface

“…It has been proposed that IF1 disrupts the contact between the ␤-and ␥-subunits, inhibiting F 1 ATPase function (12). In addition, the binding of IF1 protein to ATP synthase depends on pH (13), with a pH of 6.5 or below favoring a stable complex with the enzyme (14). The ability of IF1 to inhibit ATP hydrolysis is well documented, but its role in the synthesis of ATP has been unclear (15)(16)(17).…”