title: Protein inhibitor of mitochondrial ATP synthase: relationship of inhibitor structure to pH-dependent regulation.
abstract: In the absence of an electrochemical proton gradient, the F1 moiety of the mitochondrial ATP synthase catalyzes the hydrolysis of ATP. This reaction is inhibited by a natural protein inhibitor, in a process characterized by an increase in ATPase inhibition as pH is decreased from 8.0 to 6.0. In order to gain greater insight into the molecular and chemical events underlying this regulatory process, the relationships among pH, helicity of the inhibitor protein, and its capacity to inhibit F1-ATPase activity were examined. First, peptides corresponding to four regions of the 82-amino-acid inhibitor protein were chemically synthesized and assessed for both retention of secondary structure, and capacity to inhibit F1-ATPase activity. These studies showed that a region of only 24-amino-acid residues, from Phe 22 through Len 45, accounts for the inhibitory capacity of the inhibitor protein, and that retention of native helical structure in this region is not essential for inhibition. Second, three mutants (33P34, 39P40, and 43P44) of the intact inhibitor protein were prepared in which a proline residue was inserted within the inhibitory region to disrupt native helical structure. The secondary structures and inhibitory capacities of these mutants were analyzed as a function of pH. These studies revealed that, despite the initial loss of helical structure within the inhibitory region due to proline insertion, a further loss of helical structure is required to modulate inhibitory activity. These results suggest that a loss of helical structure outside the inhibitory region correlates with an increase in inhibitory capacity. Finally, two separate mutants (H48A and H55A) were prepared in which a conserved histidine residue in the wild-type inhibitor protein was replaced with an alanine. The secondary structures and inhibitory capacities of these mutants were also investigated as a function of pH. Results indicated that, although histidine residues do not directly affect the inhibitory capacity of the protein, they are important for maintaining the inhibitor protein in an inactive form at high pH. Furthermore, these results show that loss in helical structure, although correlated with an increase in inhibitory capacity, is not essential for this function. These novel experiments are consistent with a model in which the inhibitor protein is envisioned as consisting of two regions, an inhibitory region and a regulatory region. It is suggested that reduction of pH allows for the protonation of a histidine residue blocking the interaction between the two regions, thus activating the inhibitory response. The pH reduction also correlates with a partial unfolding of the protein that may either cause or result from the loss of interaction between the two helices. This unfolding may be necessary for further optimization of inhibitor function.
authors.affiliation: Laboratory for Molecular and Cellular Bioenergetics, Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Lebowitz
authors.forename: P L
authors.initials: PL
authors.lastname: Pedersen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011392:Proline / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D006639:Histidine; D011392:Proline; D006180:Proton-Translocating ATPases