Characterization of CopA, a P1B-type Copper(I)-ATPase.

摘要

Copper plays important roles in the cell, but because of its ability to disrupt cell processes, copper localization and concentration must be tightly controlled in the cell. A variety of protein trafficking machinery, including metallochaperones and P-type ATPases mediate resistance to copper. P-type ATPases are metal specific pumps that play a central role in resistance to heavy metal toxicity, including copper. These proteins couple the hydrolysis of ATP to substrate translocation across the membrane, thus pumping copper out of the cell. Mutations in the human versions of these copper pumps lead to Wilson's and Menkes diseases, characterized by mishandling of copper in the cell. In general, P-type ATPases turnover by alternating between high and low affinity substrate states, described by the Post-Albers cycle. A detailed mechanism by which this copper P-type ATPases tightly coordinate copper and move it across the membrane is currently lacking. The residues that form the binding site, making the pump selective for specific ions and how this site changes during the translocation cycle is poorly understood. In addition, the role that MBDs play in the translocation of copper is ambiguous. Finally, there have been reports of importing P-type copper ATPases; however, little biochemical evidence has been published to support these claims.;Structural characterization of this membrane protein would clarify the mechanistic questions posed by these pumps. Crystallization of soluble domains were carried out to understand the roles of the metal binding and actuator domains. The structure function relationship of these domains was further characterized. Crystallization of membrane proteins is a challenging endeavor, a process expedited by unique approaches to the crystallization problem. Multiple Cu+ P-type ATPase homologs were targeted, and low resolution diffraction data were obtained. Finally, in the course of these studies, a P-type ATPase with a unique activity profile was isolated from Methylococcus capsulatus (Bath) and biochemically characterized.