Bound metals are present in approximately one-third of all proteins and, as such, these micronutrients represent a central component in all biological systems. Copper is directly involved in many human physiological processes and protects cells from potentially mutagenic DNA damage by free radicals. Copper deficiency has been implicated in anemia, cardiovascular abnormalities, neurodegeneration, tumorigenesis, angiogenesis, and neutropenia (a common toxicological consequence of chemotherapy). The integral membrane protein Ctrl is responsible for the cellular uptake of copper. Unexpectedly, hCtrl is also responsible for uptake of platinum cytotoxic anticancer drugs such as cisplatin, and thus presents a possible target for the design of related drugs with improved uptake properties, hCtrl is 190 amino acids in length, contains three predicted transmembrane helices, and is most likely physiologically functional as a trimer. Ctrl possesses a novel fold, and its molecular basis of function is not known. Human Ctrl (hCtrl) has been cloned into the methanotropic yeast Pichia pastoris, multiple constructs have been designed and built, expression has been optimized, and reproducible high-level expression has been achieved. A robust two column purification method has been developed that enables the facile generation of multi-milligram quantities of highly-pure hCtrl.
The aims of this R21 application, submitted under Program Announcement PA-03-100 'NIGMS Exploratory Studies for High Impact/High Risk Research', are to: 1) produce multimilligram quantities of purified hCtrl, and 2) conduct systematic three-dimensional crystallization experiments with hCtrl and hCtrl complexes. The goal of this proposal is to obtain crystals sufficient for structure determination of hCtrl by x-ray crystallography. The longer-term goals of this effort are to conduct detailed structure-function studies of hCtrl, to critically evaluate the utility of the hCtrl structure for design of novel platinum anticancer drugs, and to extend our research to the structural biology of copper homeostasis.
