Structural Biology

Molecular mechanisms of anthrax toxin assembly and transmembrane protein transport

Proteins rarely carry out cellular function proximal to ribosome of their synthesis; thus protein transport represents a crucial step in the central dogma of biochemistry. At least half of all eukaryotic proteins must transverse a membrane, which requires catalysis by protein channel nanomachines, usually involving protein unfolding, refolding, and thus energy. Anthrax toxin is a useful model system for understanding so-called “protein translocation,” enabling the molecular assembly, unfolding, membrane translocation, and refolding steps to be studied structurally, biophysically, and biochemically.

Dissertation by Geoffrey K. Feld, Ph.D. (2012) University of California, Berkeley. Advisor: Prof. Bryan A. Krantz, Ph.D.

Cover, March 2010 Nature Struct Mol Biol.
Anthrax toxin Protective antigen, 1.45-A resolution (PDB: 3TEW)
Protein translocation by a proton-driven engine (credit: Protein Science)

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 determined 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 (the α clamp). The α clamp is critical for toxin assembly, substrate binding, unfolding, and translocation through a nonspecific binding mechanism.

Feld GK*, Thoren KL*, Kintzer AF, Sterling HJ, Tang II, Greenberg SG, Williams ER, Krantz BA. (2010) Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers. Nature Struct Mol Biol.17:1383-1390. NSMB Article of the Month (Oct)

  • PDB: 3KWV (Prechannel core of Anthrax lethal toxin complex: PA octamer bound to Letha Factor translocation domain [LFn])

At the beginning of my graduate studies, our lab (Prof. Bryan Krantz, then at UC Berkeley) discovered that anthrax toxin PA could form an octamer, in addition to the well-described heptamer. The octamer is physiologically relevant, being the predominant form at body temperature and pH, and a significant minor fraction when combined with LF in vitro.

Kintzer AF, Thoren KL, Sterling HJ, Dong KC, Feld GK, Tang II, Zhang TT, Williams ER, Berger JM, Krantz BA. (2009) The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes. J Mol Biol.392(3):614-629. 

The ability of PA to form two physiologically-relevant 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.

Feld GK, Kintzer AF, Tang II, Thoren KL, Krantz BA. (2012) Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen. J Mol Biol. 415:159-174.   

  • PDBs: 3TEW (Anthrax protective antigen [PA], highest resolution available 1.45A); 3TEX (PA low pH); 3TEY (PA disulfide mutant); 3TEZ (PA disulfide mutant plus linker)

The above concepts of anthrax toxin assembly and transport and how they relate to similar systems across biology were also comprehensively reviewed, including a proposed model for helical, nonspecific binding, Brownian ratchet-based protein translocation.

Feld GK, Brown MJ, Krantz BA. (2012) Ratcheting up protein translocation with anthrax toxin. Protein Sci. 21:606-624.

Enabling membrane protein structural biology with X-ray lasers

Membrane proteins are notoriously difficult to crystallize and determine structures, yet they represent most of the human drug targets. Proteins that catalyze photon-driven biochemistry (e.g., photosynthesis) typically undergo important structural changes on very fast timescales that pose challenges for structural biologists. A better understanding of these processes at the atomic level will inform on therapeutic discovery and bio-mimicking materials that will help sustain energy production for future generations. X-ray lasers (a.k.a. X-ray free electron lasers or XFELs) are multidisciplinary “Big Science” projects at national labs in the United States (the LCLS and forthcoming LCLS II at Stanford’s SLAC laboratory), Europe (the SwissFEL at Swizerland’s Paul Scherer Institute and the European XFEL in Hamburg, Germany), Japan (SACLA at Spring8 in the RIKEN Institute), and South Korea (PAL-XFEL).

Membrane protein structures collected at room temperature (XFEL) differ from those collected at cryogenic temperatures (courtesy of Curr. Op. Struct. Biol.
Experimental setup of the fixed target approach at an XFEL (courtesy of Sci. Rep.)
XFEL diffraction from purple membrane on a low-Z polymer EM grid

We were invited to write a comprehensive review of the state-of-the-art (as of 2014) in X-ray laser biology with a focus on membrane proteins in Current Opinion in Structural Biology.

Feld GK & Frank M. (2014) Enabling membrane protein structure and dynamics with X-ray free electron lasers. Curr. Op. Struct. Biol. 27:69-78.

A major challenge in doing structural biology with X-ray lasers is the so-called “diffract-before-destroy” mechanism of the technique. In conventional X-ray crystallography, a protein crystal is rotated in the X-ray beam as data are being collected, which spreads out the “spots” and supports complete data collection to reconstruct the biomolecule under investigation. In contrast, each pulse of the XFEL turns the irradiated sample into plasma, meaning the sample must be constantly replenished with each successive X-ray shot. Most methods result in an excess of precious protein crystal sample being consumed with a low “hit-rate,” that is, few of the nano- or microcrystals interact with the FEL X-rays. Complicating the problem are the “pump-probe” experiments, in which laser light (usually at visible or UV wavelength) is used to activate (“pump”) a photon-sensitive molecule (e.g., Photosystem II), and then diffraction from the XFEL is used to determine the structure (“probe”) and make “molecular movies.”

To address these sample consumption and logistical issues, our team at Lawrence Livermore National Laboratory spearheaded a worldwide collaboration of SLAC, PSI, PNNL, and academic scientists to develop “fixed-target” sample introduction approaches, whereby slurries of microcrystals were added to silicon nitride wafers to maximize hit rate and retain sample. In this manner, we reported the first room-temperature XFEL diffraction studies of 2D protein crystals using purple membrane as a model system.

Pedrini B, Tsai CJ, Capitani G, Padeste C, Hunter MS, Zatsepin NA, Barty A, Benner WB, Boutet S, Feld GK, Hau-Riege SP, Kirian RA, Kupitz C, Messerschmitt M, Ogren JI, Pardini T, Segelke BW, Williams GJ, Spence JCH, Abela R, Coleman MA, Evans JE, Schertler GFX, Frank M, Li XD. (2014) 7 Å resolution in protein 2D-crystal X-ray diffraction at LCLS. Philos Trans R Soc B. 369(1647):20130500

To help unify the methods of probing 2D biomolecules with cryoelectron microscopy and X-ray lasers, our team also developed a system of mounts so that XFEL diffraction could be measured from conventional EM grids, as well as a series of polymer grids and wafers to improve diffraction data and enable high-throughput data collection.

Feld GK*, Heyman M*, Benner WH, Pardini T, Tsai CJ, Boutet S, Coleman MA, Hunter MS, Li X, Messerschmidt M, Opathalage A, Pedrini B, Williams GJ, Krantz BA, Fraden S, Hau-Riege SP, Evans JE, Segelke BW, Li, XD, Frank M. (2015) Low-Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography. J Appl Cryst. 48:1072-1079. 

Structure and function of a novel protein that enables a biothreat agent to infiltrate cells

Feld GK, El-Etr S, Corzett MH, Hunter MS, Belhocine K, Monack DM, Frank M, Segelke BW, Rasley A. (2014) Structure and function of REP34 implicates carboxypeptidase activity in Francisella tularensis host cell invasion. J Biol Chem. 289:30668-79.

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Here, we characterize the cellular and molecular function of REP34, a rapid encystment phenotype (REP) protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with a novel typology and close structural homology to the metallocarboxypeptidase family of peptidases. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

  • PDBs: 4K0K0 (Rapid Encystment Phenotype Protein 34 KDa [REP34] punitive virulence factor from Francisella tularensis)
  • 4P5P ((Rapid Encystment Phenotype Protein 24 KDa [REP24] punitive virulence factor from Francisella tularensis)
REP34 (PDB: 4K0K0)

Popular press articles on “Rabbit Fever” research

Structure determination of REP34 from F. tularensis

Presentation of the above REP structure-function study at the 2014 Biophysical Society meeting in San Francisco, CA prompted several adoptions of the narrative in the popular press, including a short segment on National Public Radio’s Science Friday with quotes from Geocyte founder, Geoffrey Feld.

Describing a novel protein involved in pathogen response to host-initiated oxidative stress

Appel MD*, Feld GK*, Wallace BW, Williams RS. (2016) Structure of the sirtuin-linked macrodomain SAV0325 from Staphylococcus aureus. Protein Sci. 25:1682-1691.

Cells use the post-translational modification ADP-ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon-encoded mono-ADP-ribosylation cycle mediates response to host-induced oxidative stress. Here we report the crystal structure of the sitruin-linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75-Å resolution. The monomeric SauMacro bears a previously unidentified Zn2+-binding site that putatively aids in substrate recognition and catalysis. Structural features of the enzyme further indicate a cleft proximal to the Zn2+ binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target.

  • PDB: 5KIV (sirtuin-linked macrodomain SAV0325 from Staphylococcus aureus [SauMacro])
SauMacro (PDB: 5KIV)

Intact protein complexes measured by nanoESI-ToF mass spectrometry

Sterling, HJ, Kintzer AF, Feld GK, Cassou CA, Krantz BA, Williams ER. (2012) Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations. J Am Soc Mass Spectrom. 23:191-200. JASMS Article of the year (2012)

Sterling HJ, Daly MP, Feld GK, Thoren KL, Kintzer AF, Krantz BA, Williams ER. Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions. J Am Soc Mass Spectrom. 21(10):1762-1774.

Shulami S, Zaide G, Zolotnitsky G, Langut Y, Feld G, Sonenshein AL, Shoham Y. (2007) A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus. Appl Environ Microbiol. 73(3):874-884.