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Structural and functional characterization of molecular nanomachines: principles of transition metal selectivity and transport in heavy metal P1B-type ATPases

Final Report Summary - P1BPUMPS (Structural and functional characterization of molecular nanomachines: principles of transition metal selectivity and transport in heavy metal P1B-type ATPases)

Transition metals such as copper, zinc, and cobalt play pivotal roles in biochemical processes critical for cellular metabolic activity and survival. In contrast, other transition metals such as cadmium, mercury or lead, by impairing crucial biochemical pathways, are toxic and actively removed from the cellular environment. P1B-type members of the P-type ATPase superfamily of membrane proteins evolved as an essential transport system for the selective translocation of transition metal ions across the cell membrane. These transporters utilize the energy generated by ATP hydrolysis to drive metal translocation across the membrane against the electrochemical gradient. The molecular mechanisms by which these nanomachines select transition metals and how the energy stored in ATP is converted into the directional flux of metal ions across the membrane are not completely understood.
We used a multidisciplinary structural and biochemical approach to obtain a molecular knowledge of the structure and the function of these transporters.
We have successfully identified conditions for recombinant expression and purification wild-type and mutant prokaryotic P1B-type ATPases from different archaea and bacteria species suitable for structural and functional characterization. The obtained targets belong to different P1B-type subfamilies which show different ion selectivity patterns, including Cu+, Cu2+, Zn2+/Cd2+, Co2+ or Ni2+. Ideal targets have been selected for crystallization trials with the long term goal of determining the three-dimensional structure of pumps with different metal selectivity.
In a bioinorganic chemical approach, we focused in understanding how transition metal selectivity and oxidation state specificity are achieved.
The following goals have been addressed:
i) Determine the metal selectivity on P1B-type ATPase exporters in detergent micelles and lipid bilayers upon reconstitution in proteoliposomes.
ii) Characterize the catalytic properties of P1B-type ATPase with exhibiting different metal selectivities.
iii) Investigate whether P1B-type ATPases are uniporters or antiporters, and/or electrogenic.
iv) Determine the number and coordination environment of transmembrane metal binding sites (TM-MBS), which are essential for substrate selectivity and transport to understand the bioinorganic chemistry of metal selection and translocation.
We characterized the metal binding properties of wild-type proteins and mutants. By using a panel of spectroscopic techniques including electronic absorption, electron paramagnetic resonance (EPR) and X-ray absorption spectroscopies (XAS) we have determined models of the transmembrane metal binding sites in Cu+, Zn2+/Cd2+ and Cu2+ ATPases. The conducted studies have revealed differences in the nature of the coordinating ligands, the geometry of metal binding in the TM-MBS, and the coordination chemistry underlying metal translocation.
We have also successfully reconstituted the purified proteins in artificial lipids bilayers (proteoliposomes) and established protocols for monitoring real-time metal and potential counter-ion transport using metal- or pH dependent fluorescent probes. With this information we are revealing the molecular principles underlying metal binding selectivity and transport in this class of transporters, providing new highlights into the bioinorganic chemistry involved in metal transport across biological membranes.
The knowledge gained in the project, on one hand, deepened our understanding of the structure and function of this class of membrane ATP pumps, and, on the other, provide the bases for the future rational design of modulators of P1B-type ATPase activity.