A-ATPASE SUBUNITS E AND G

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proton translocating ATPases are membrane bound complexes that are central to biological energy conversion and use a rotary catalytic mechanism. Vacuolar ATPases are versatile proton pumps that use the energy of ATP to build up proton gradients across internal membranes of eukaryotic cells. These proton gradients are essential for intracellular pH homeostasis and for transport processes, such as neurotransmitter uptake into synaptic vesicles and bone resorption by osteoclasts. The related A-type ATPases occur in the membranes of archaeal and certain bacterial cells where they are mostly responsible for ATP synthesis. Although simpler by design than their eukaryotic counterparts, A- ATPases are even more versatile molecular machines in that they are reversible and can function both as ATP synthases and as proton pumps in dependence of cellular requirements. We have analysed the intact 680 kD A-ATPase from T. thermophilus by electron microscopy and performed a 3D reconstruction which provided us with an electron density to 23 ¿ resolution. We are using this electron density to build a pseudo-atomic model of the complex by docking in high-resolution X-ray structures. The major part of our model that is still missing is the peripheral stator, which is build up by a complex of subunits E and G. The stator connects the nucleotide binding subunits and the non-rotating part of the proton channel and counteracts rotation. It is also essential for the assembly and disassembly of the complex, which is believed to have regulatory function. So far we have collected a native data set to 2.4 ¿ resolution, but we still need MAD or SAD data of selenomethionine containing crystals. The crystals belong to space group P21212 with unit cell dimensions of a=102 ¿, b=208 ¿, c=37 ¿ and most likely two copies of EG per asymmetric unit. We have modified the sequence of subunit E by site directed mutagenesis, resulting in 4 methionines within a total of 290 residues per EG complex.