Grant: Operating Grant

Anthrax toxin is comprised of two toxic enzymes and a third protein, PA, which assembles into a nanomachine that delivers the toxic enzymes to the interior of human cells. We are studying two critical steps in the assembly of this translocation machine. The first step we will study is the binding of PA to receptors on human cells - this allows the toxin to target susceptible cells. We have discovered that the extracellular part of the receptor exists in two different conformations and that only one of these conformations is able to bind toxin. Furthermore, we have shown that another region of the receptor, located inside the cell, is able to bind the cytoskeleton protein actin. It is the binding of actin to the receptor that switches the receptor from the conformation that binds toxin to the conformation that does not bind toxin. We propose to study different parts of the receptor to determine how the binding to actin inside the cell is able to change the conformation of the receptor outside the cell. The second step in the assembly process we will study is the insertion of the toxin into the cell membrane. This is an essential event because membrane insertion forms a hole through which the toxic enzymes pass to the cell interior. The receptors stabilize the toxin until the toxin reaches an acidic region of the cell; the acidity breaks an interaction between the toxin and its receptor, which allows membrane insertion. We will study how the receptor controls insertion of the toxin and the interactions that allow insertion to occur in a highly efficient manner. The work described in this proposal elucidates fundamental aspects of protein function and regulation. The information obtained will be useful for the engineering of protein translocation nanomachines and will impact drug delivery systems.