The H+-transporting F1F0 ATP synthases of oxidative phosphorylation in mitochondria and bacteria are very similar. Rotation of subunit gamma within the core of the alpha-3-beta-3 hexamer of F1 drives ATP synthesis by a unique rotary catalytic mechanism. H+ transport through transmembrane F0 drives rotation of an oligomeric ring of c subunits connected with gamma, and results in ATP synthesis in catalytic sites at the alpha-beta interface. A stator complex of F0 subunits a and b and F1 subunit delta extends from the membrane to the top of the F1 molecule and holds alpha-3-beta-3 fixed, relative to the membrane, allowing the c-gamma complex to rotate within. The mechanism of coupling H+ transport and c-ring rotation is poorly understood. The structure of subunit c was solved by solution NMR and the c-ring has been modeled. Biochemical evidence indicates that one of the helices of subunit c, which resides at the interface with subunit a, rotates between two different conformations. The concerted rotation of helices at the subunit a-c interface is proposed to mechanically drive the stepwise movement of the c-ring. This proposal focuses on the structure of subunit a, with the ultimate goal of defining its role in coupling H+ transport to c-ring rotation. The global fold and packing of subunit a in native Escherichia coli membranes will be determined by cross link analysis. Aqueous access pathways in subunit a mediating H+ transport from membrane surfaces to the H+ binding site in subunit c will be defined, and the mechanism of gating H+ access to the two sides of the membrane probed. Simultaneously, we will attempt to determine the solution structure of purified subunit a by NMR. Initially, the global fold of the purified protein in solution will be compared to that in the membrane using spin-labeled protein to establish appropriate solution conditions. Ultimately, we hope to define an atomic resolution structure that can be used in mechanistic studies. The ATP synthase is central to cellular function--it makes the ATP. Abnormalities in the enzyme lead to human disease. Closely related enzymes are responsible for vesicular acidification in human cells, and work by a similar rotary mechanism. The principles by which this enzyme works may provide fundamental insights into other transport problems in biology and medicine.
Abnormalities in the mitochondrial F-type ATP synthase do lead to human disease; e.g. Leigh'sdisease with defects in subunit a and Batten's disease where subunit c aggregates in diseasedneurons. The F-type ATPase is also found in multitudes of pathogenic bacteria and is a potentialdrug target. Subunit c was recently shown to be the target of a new class of anti-TB drugs(arylquinolines) that seem to be specific to Mycobacterium tuberculosis with neglible effects onmammalian ATP synthase. The drug binding site is near the essential H+-translocating Asp/Gluand the hydrophobic sequence in this region of the protein is implicated in drug resistance. Ourstudies on on the role of subunit a in providing aqueous access and inhibitor access to this regioncould contribute to better drug targeting methods. Closely related V-type ATPases are responsiblefor vesicular acidification in human cells; and work by a similar rotary mechanism. What we findhere will clearly be applicable to this widespread family of enzymes; which function in a diversityof physiological roles; from intracellular membrane trafficking to bone reabsorption and urinaryacidification. A mechanistic understanding of F1Fo ATP synthase may well provide insights intoelucidation and treatment of diseases caused by defects in the V-type family of enzymes; wherehomologues of subunits a and c clearly have similar functions to that in E. coli.
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