UC Berkeley

Because proteins carry out their cellular functions in specific locations, protein transport represents a crucial step in the central dogma of biochemistry. Transport across a membrane barrier requires energy; therefore, nature has provided a class of molecular machines known as translocases that catalyze protein translocation. Often, these machines contain channels that are too narrow to transport a folded protein; thus, substrate unfolding adds another layer of complexity to the poorly understood transport mechanism. The anthrax toxin system represents a useful model for elucidating the mechanisms of translocation-coupled protein unfolding, both in terms of structure and function.

In order to understand the mechanism by which an unfolding machine interacts with its substrate, the X-ray structure of an anthrax protective antigen (PA) oligomer prechannel was solved in complex with the amino-terminal PA-binding domain of the substrate lethal factor (LF_N). The structure revealed how PA interacts with unfolded polypeptide via a hydrophobic cleft we call the &alpha clamp. Working with my colleague, Katie Thoren, we show how the &alpha clamp is critical for toxin assembly, substrate binding, unfolding, and translocation through a nonspecific binding mechanism.

The recent discovery that PA can form two oligomeric states exemplifies the complexity of multimeric protein nanomachine assembly. In order to better understand the molecular mechanism of heterogenous assembly, a series of PA constructs with varying length crosslinks was produced, their structures were solved to high resolution, and their assembly products were analyzed by electron microscopy. The flexibility of PA's receptor-binding domain (D4) relative to the main body of the protein provides a mechanism for controlling oligomeric stoichiometry, whereby D4 can adopt a pro-heptamer or pro-octamer conformation.

Finally, an overall model for protein translocation is presented, based on data discussed herein and other recent studies involving anthrax toxin transport.