Research project
The understanding of metal transport in human cells is critical for finding rational treatments to many debilitating human conditions. Copper is an important metal obtained from the diet that is required in many key enzymes but is toxic when free in solution. In addition, copper ions have been found in amyloids and in tumors.
Despite structural, genetic and biochemical data on copper-transfer proteins from humans and many other organisms, mechanistic aspects of the reactions of these proteins are not yet elucidated. Inside human cells, the copper chaperone Atox1 delivers copper to metal-binding domains of Wilson and Menkes proteins in the Golgi, which then load the metal onto target protein and enzymes, such as ceruloplasmin which is a plasma protein important for iron metabolism. In this project, we will address the molecular mechanisms and the proteins involved in this pathway. Not only will this information provide a basic understanding of the rules that drive human copper transport, but it will also help reveal how mutations lead to Wilson disease and other disorders involving copper imbalance.
Copper (Cu) is an essential element for the survival of most living organisms. To avoid toxicity, a group of copper-carrier proteins called metallochaperones bind Cu(I) via cysteine residues and transfer the metal directly to specific partner proteins. In the human secretory pathway, the cytoplasmic copper chaperone, Atox1, delivers Cu to the P-type ATPase, Wilson disease protein (WND) located in the trans-Golgi network. WND uses ATP hydrolysis (in N- and P-subdomains) to transport Cu from its cytoplasmic metal-binding domains (WND1-6) to the lumen of the secretory pathway, where most copper-dependent enzymes acquire Cu. To obtain a molecular understanding of human copper metabolism and its disorders, the biophysical properties and physiological functions of the Cu transport proteins and disease-causing variants must be examined. Here, we will use a set of biophysical methods to address Cu transfer mechanisms for human proteins involved in the Golgi transport system. Aim 1. Where in WND does Atox1 deliver Cu and why? We hypothesize that Cu is delivered only to WND2 or WND4 due to unique conformation/dynamics of their metal-binding cysteines. Efficiency of Atox1-mediated Cu loading of different WND domains will be tested. Unfolding experiments of the six WND domains will probe if low stability, allowing for conformational flexibility, is necessary for Cu uptake. Molecular dynamics (MD) simulations will assess structural and dynamic differences among the six WND domains. Aim 2. Does the transient complex involve Cu sharing and conformational changes? We hypothesize that copper transfer involves a trigonal Cu coordination between partially-unfolded proteins. Real time Cu transfer will be monitored to identify rate-limiting steps and mechanisms. Separate Cu dissociation and unfolding tests will reveal if such processes are involved in transfer. Simulations will pinpoint Cu geometry in the transient hetero-complex. Aim 3. Is further Cu transfer regulated by long-range WND interactions? We propose that Cu is transferred between selected WND domains facilitated by conformational changes and N-subdomain interactions. Unfolding of multi-domain constructs will be probed as a function of Cu to reveal if they act as globular domains or “beads on a string”. Internal Cu transfer between various WND domains will be tested as well as the ability of the N-subdomain to interact with WND16.
