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UC Berkeley UC Berkeley Electronic Theses and Dissertations UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Molecular Mechanisms of Anthrax Toxin Assembly and Transport Permalink https://escholarship.org/uc/item/3hm635k0 Author Feld, Geoffrey Keith Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Molecular Mechanisms of Anthrax Toxin Assembly and Transport by Geoffrey Keith Feld A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Bryan Krantz, Chair Professor David Wemmer Professor Susan Marqusee Spring 2012 Molecular Mechanisms of Anthrax Toxin Assembly and Transport ©2012 Geoffrey Keith Feld Abstract Molecular mechanisms of anthrax toxin assembly and transport by Geoffrey Keith Feld Doctor of Philosophy in Chemistry University of California, Berkeley Professor Bryan Krantz, Chair 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 (LFN). The structure revealed how PA interacts with unfolded polypeptide via a hydrophobic cleft we call the α clamp. Working with my colleague, Katie Thoren, we show how the α 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-PA7 or pro-PA8 conformation. Finally, an overall model for protein translocation is presented, based on data discussed herein and other recent studies involving anthrax toxin transport. 1 Dedicated to my family. i Table of Contents List of Figures............................................................................................................................... iv List of Tables..................................................................................................................................v List of Abbreviations.....................................................................................................................vi Acknowledgements.......................................................................................................................vii Chapter 1 Ratcheting up protein translocation with anthrax toxin........................................................... 1 1.1 Nanomachines as ratchets..................................................................................................... 1 The physical environment of the cell...................................................................................... 5 1.2 Molecular mechanisms of biological nanomachines............................................................ 5 1.3 Anthrax toxin as a protein translocation model system........................................................ 7 1.4 The PA channel................................................................................................................... 11 The LF/EF binding sites........................................................................................................ 12 The φ clamp........................................................................................................................... 12 The β barrel........................................................................................................................... 14 1.5 Translocation powered by a proton gradient....................................................................... 15 1.6 Translocation-coupled protein unfolding............................................................................ 16 1.7 Processing unfolded protein................................................................................................ 17 Chapter 2 Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers............................................................................................. 19 2.1 Abstract............................................................................................................................... 19 2.2 Introduction......................................................................................................................... 19 2.3 Results................................................................................................................................. 20 Crystal structure of the PA8(LFN)4 complex......................................................................... 20 The carboxy-terminal binding subsite................................................................................... 22 The α-clamp binding subsite................................................................................................. 22 Both LF-binding subsites are critical for cytotoxicity activity.............................................. 31 The role of the α clamp in LT assembly................................................................................ 31 Mapping the LFN-binding interaction with the PA channel.................................................. 31 The α clamp possesses nonspecific binding activity............................................................. 34 ii LFN must unfold to bind the α-clamp subsite....................................................................... 36 Binding to PA induces strain and disorder into LFN............................................................. 36 The role of the α clamp in protein translocation................................................................... 42 2.4 Discussion........................................................................................................................... 42 2.5 Materials and Methods........................................................................................................ 49 Chapter 3 Domain flexibility modulates the heterogenous assembly mechanism of anthrax protective antigen................................................................................................. 56 3.1 Abstract............................................................................................................................... 56 3.2 Introduction......................................................................................................................... 56 3.3 Results................................................................................................................................. 59 Crystal structure of the PAΔMIL monomer............................................................................. 59 Ordered loop regions............................................................................................................. 61 Receptor-binding loop-helix 2α1........................................................................................... 61 Changes in orientation of PA D4.......................................................................................... 61 Crystal structures of cross-linked PA monomers.................................................................. 63 Crystal structure of PAΔMIL at pH 6.5.................................................................................... 63 The D2-D4 interface modulates the propensity of PA to form octamers.............................. 63 Correlation of D4 conformation and oligomeric heterogeneity............................................ 66 The furin-cleavage site.......................................................................................................... 66 Furin-dependent PA processing is not ANTXR2-dependent................................................ 69 3.4 Discussion........................................................................................................................... 69 Protoxin maturation by furin................................................................................................. 70 Molecular determinants of PA’s heterogenous oligomerization mechanism........................ 71 3.5 Materials and methods......................................................................................................... 72
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