Metal micronutrients, Fe, Cu, Zn, etc., play fundamental catalytic and structural functions in proteins. Proper metal levels are achieved by the coordinate action of specific chaperones and transmembrane transporters. The goal of this project is to understand the fundamental biochemistry involved in transmembrane heavy metal transport by ubiquitous P1B-type ATPases. Alkali metals (Na+, K+, Ca2+) are free (hydrated) in the cellular milieus. As such, they reversibly interact with and are transported by channels, co-transporters, and pumps. In contrast, heavy metals are bound to chaperones/chelating molecules with high association constants. This project aims to understand how heavy metals are delivered to their transporters, how they are coordinated with affinities likely higher than that of their chaperones, and how they are subsequently released from the transporter. In these studies, Archaeoglobus fulgidus CopA will be used as a model Cu+-ATPase. Experiments will be performed to test whether CopZ, the A. fulgidus Cu+ chaperone, interacts with CopA delivering Cu+ to transmembrane metal binding sites. The stoichiometry of metal binding to transmembrane sites will be established along with the atomic coordination and the arrangement of coordinating amino acid side chains. The project will also examine the structural mechanism of allosteric regulation by metal binding to cytoplasmic domains. Finally, the fourth goal of the project is to establish the complete atomic resolution structure of a Cu+ transporting ATPase. This would greatly enhance understanding the mechanism of heavy metal transport.
Broader Impact: The project will contribute to understanding the principles governing metal binding, translocation and release mechanisms of metal transporters. The research will contribute to ongoing educational activities including undergraduate teaching, curriculum development and summer research. Graduate and undergraduate students form the core of the laboratory and will perform major parts of this project. This work will also impact a close collaboration with K-12 programs at our institution. This ranges from guiding high school students working in the lab to participation in high school teachers training.
The performed studies have contributed to understand the mechanism and the function of copper transport across biological membranes. This processes are essential for the life of all organisms and the symbiosis of beneficial bacteria en their interaction with the hosts. Heavy metals like copper are toxic when free in the cell but need to be placed in the appropriate cuproenzymes. The studies have showed how copper is bound by the transporter and subsequently translocated across biological membranes. The project shoed how chaperone proteins transfer the metal to the transporter and then the transporter delivers to another chaperone in the neighboring compartment. Details of how the metal is coordinated and how ligating groups are exchanged were determined. The exquisite selectivity in the interaction of these proteins was also described. The studies produced data leading to novel paradigms to explain how cells handle copper. Results were reported in many publications that are highly referenced in the field, showing the importance of our observations. A number of post-doctoral fellows, graduate students and undergraduate students participate in these studies. These have now continue their scientific either in academic or industrial settings. Members of the research team were also involved in various community activities ranging from judging High school science fairs and directing high school interns during the summers, to mentoring younger colleagues and doing general public presentations.
