Cryo-Electron Microscopy Structure Determination of the Anthrax Toxin Protective Antigen Bound to its Lethal and Edema Factors
AdvisorKrantz, Bryan A.
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AbstractProtein translocation is an essential function within all living cells. Translocons are dedicated protein translocation machinery, responsible for the unfolding and translocation of proteins. Due to the thermostability of most proteins in their native states, these translocons utilize various different forms of energy to drive the translocation of their substrates. This process is mediated by polypeptide clamps responsible for catalyzing the unfolding and translocation of the protein. Using lipid nanodiscs and cryo-electron microscopy (cryoEM), we have determined structures of heptameric anthrax lethal toxin and edema toxin channels to 4.6 and 3.2-Å resolution, respectively. Additionally, using cryoEM we have determined the first atomic structures of PA8 prechannel bound to full-length EF and LF to 3.3 and 3.7-Å resolution, respectively. In this pre-translocation state, the first α helix and β strand of LF and EF unfold and the α clamp, which resides at the interface of two PA subunits. The α clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins, supporting previous work indicating that the α-clamp engages substrate α-helices repeatedly during translocation. A PA loop in the binding interface is displaced between the prechannel and channel. This results in the loss of a salt bridge and leading to the weakening of the binding interface prior to translocation in the PA7EF structure. Lastly, EF undergoes a largescale conformational rearrangement when forming the complex with PA, compared the solution structure of EF bound to calmodulin. Recruitment to the PA prechannel exposes an originally buried β strand and enables domain organization of EF. Many interactions are formed on domain interfaces in both PA prechannel-bound EF and LF, leading to toxin compaction prior to translocation. This work has resulted in the first structures of PA bound by edema factor, as well as the first structures of PA bound to a full-length substrate. These structures have provided insight into important biophysical steps occurring in preparation for translocation They reveal structural plasticity within the binding α-clamp binding site, allowing the translocating substrate to be engaged multiple times. This provides a greater understanding of how anthrax toxin can invade the host cytosol.
University of Maryland, Baltimore