P-type ATPases & Cu+ transport by CopA
|Copper is essential to cells as a cofactor for a wide variety of enzymes. If not carefully controlled, however, Cu can be toxic due to its redox potential and its ability to produce free radicals. As a result, a system of pumps, transporters and metallochaperones have evolved to control the delivery and distribution of Cu. In general, intracellular Cu-scavengers, such as metallothioneins, ensure that there is a vanishingly low concentration of free Cu in the cytoplasm and metallochaperones are used to carry the Cu through the cytoplasm and to deliver it to specific targets. In mammals, Ctr1 is a secondary transporter on the cell surface that facilitates entry into the cell and two transmembrane ATPases, ATP7A and ATP7B, that pump Cu across the endoplasmic reticulum and plasma membrane, respectively. Mutations in ATP7A gives rise to Menkes disease, resulting from insufficient delivery of Cu in brain and other tissues, whereas mutations to ATP7B are responsible for Wilson's disease, where Cu overload is responsible for liver and brain dysfunction. ATP7A, and ATP7B are homolgous to CopA from bacteria and belong to the P-type ATPase family comprising ATP-dependent, transmembrane ion pumps. P-type ATPases can be divided into five subgroups, PI -PV, and include Ca2+-ATPase, Na+/K+-ATPase, H+-ATPases and well as lipid flippases. The PIB subfamily contains CopA, ATP7A, and ATP7B along with transporters of a diverse array of transition and heavy metal ions, such as Cu+, Cu2+, Zn2+, Cd2+, Co2+, and Pb2+.|
CopA and its relatives in the PIB family, are distinguished from other P-type ATPases by having only eight transmembrane helices and by bearing one or more N-terminal metal-binding domains (NMBD). These NMBDs are homologous to the soluble metallochaperones. Both NMBDs and metallochaperones bind Cu+ with high affinity via CxxC sequence motifs. Although it has been postulated that NMBDs are mediate transfer of Cu from the metallochaperones to transport sites, there is increasing evidence that these domains are instead involved in autoregulation and, in the case of ATP7A, in targeting the molecule to the basolateral membrane. We are studying the structural basis for this autoregulation by electron crystallography.
Tubular crystals were grown for constructs of CopA lacking either the C-terminal metal binding domain or both the C-terminal and N-terminal metal binding domains. Images below show a low magnification overview of negatively stained tubes and a high magnification image of a frozen tube. The latter are used for helical reconstruction using Fourier-Bessel methods. The resolution of our best reconstruction is ~10 A.
The 3D reconstructions reveal the helical arrangement of CopA molecules within a cylindrical bilayer. In the best ordered crystals, the cytoplasmic domain of CopA faces the inside of the tube (contour plot below left). A model for the atomic structure of CopA was built by homology to other P-type ATPases and fit to the 3D structure of one molecule of CopA (below middle). Computational docking was used to locate the most probable site of interaction between the N-terminal metal binding domain and the cytoplasmic domains of CopA (below right). This site is consistent with an earlier reconstruction of CopA, which show extra density in this location. We believe that this metal binding domain is playing a regulatory role on the activity of CopA, keeping the pump inactive in the absence of Cu+ and thus preventing the unproductive use of ATP unless Cu+ is actually present.
Contour plot of a section through the CopA reconstruction, showing a circular lipid bilayer with cytoplasmic domains of CopA facing the inside of the tube. Black contours are positive density and green dashed contours are negative.
Fitting of a homology model for CopA to the density from the EM reconstruction. The resolution is ~10A and there is evidence of secondary structure in the density map.
Computational model for the CopA structure showing the predicted site of the metal binding domain (purple) next to the cytoplasmic domains. Transmembrane domains are colored blue.