Molecular mechanism of copper transporting ATPases

Copper ATPases are key members of P1B-type ATPases that pump heavy metal ions (Cu, Ag, Zn, Cd, Co, Pb) across cell membranes with energy derived from ATP hydrolysis. Copper ATPases pump Cu ions specifically and play vital roles in supplying essential copper to the required enzymes and in removing toxic or excess copper from cells.

Mal-functions of human copper pumps ATP7A or ATP7B disrupt copper homeostasis and this is associated with fatal disorders including Menkes and Wilson diseases and with a number of neurodegenerative diseases including Alzheimer’s, Parkinson’s and motor neuron diseases.

Hundreds of mutations in ATP7A and ATP7B have been identified and many appear to be associated with disease states. However, the molecular consequences of most of these mutations are not known, primarily due to the complexity of these copper ATPases and to the lack of effective analytical approaches for functional analysis. Over the years, in vivo protein trafficking in response to varying copper levels and in vitro ATPase activity based on detection of inorganic phosphate product are the main functional assays. However, neither is able to distinguish the functional differences between most protein variants sensitively and reliably.

Starting with a copper ATPase from E. coli, we have recently developed robust assays for Cu ATPase activity and for the correlated vectorial copper transport function. The former employed 1H NMR detection of ATP hydrolysis directly and the latter detected Cu uptake by biomimetic cells of synthetic giant unilamellar vesicles (GUVs) with embedded Cu ATPases. These assays provide an unprecedented set of quantitative tools that, with quantitative copper probes, will explore the molecular mechanisms of copper ATPases. In particular, the two essential human copper pumps, ATP7A and ATP7B, will be targeted.

References:

(1) Barnham, K. J.; Bush, A. I., Curr. Opin. Chem. Biol. 2008, 12, 222-8.

(2) Wijekoon, C.J.K.; Udagedara, S. R.; Knorr, K. L.;  Dimova, R.; Wedd, A. G.; Xiao, Z., J. Am. Chem. Soc. 2017, DOI: 10.1021/jacs.6b12921.