Selection of the division site in

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of approximately 10(6) M-1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATP gamma S and GTP gamma S and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.

We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-Å and 28-Å resolution, respectively. The structures were mushroom-shaped objects with a 125-Å-diameter cap and a 100-Å-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.

A major goal in understanding the pathogenesis of the anthrax bacillus is to determine how the protective antigen (PA) pore mediates translocation of the enzymatic components of anthrax toxin across membranes. To obtain structural insights into this mechanism, we constructed PA-pore membrane complexes and visualized them by using negative-stain electron microscopy. Two populations of PA pores were visualized in membranes, vesicle-inserted and nanodisc-inserted, allowing us to reconstruct two virtually identical PA-pore structures at 22-Å resolution. Reconstruction of a domain 4-truncated PA pore inserted into nanodiscs showed that this domain does not significantly influence pore structure. Normal mode flexible fitting of the x-ray crystallographic coordinates of the PA prepore indicated that a prominent flange observed within the pore lumen is formed by the convergence of mobile loops carrying Phe427, a residue known to catalyze protein translocation. Our results have identified the location of a crucial functional element of the PA pore and documented the value of combining nanodisc technology with electron microscopy to examine the structures of membrane-interactive proteins.electron microscopy | protective antigen | normal mode flexible fitting D etermining the structures of the membrane-inserting components of protein toxins that act within cells is critical for understanding how these toxins translocate their enzymatic cargoes across membranes. The three proteins that comprise anthrax toxin combine to form two binary toxins that contribute to the lethality accompanying infections by Bacillus anthracis. Protective antigen (PA), after binding to a cell-surface receptor and being proteolytically activated, oligomerizes to form a heptameric prepore. The prepore binds the enzymatic lethal factor and/or edema factor proteins, and, after the complexes are trafficked to the endosome, the acidic environment induces the prepore to form a pore in the membrane and to translocate the enzymatic moieties to the cytosol (1). A primary goal for understanding this process is to determine the mechanism by which the PA pore functions as a pH gradient-driven translocation machine to transport the enzymatic toxin components across membranes. Even though the x-ray crystallographic structure of the heptameric PA prepore was solved in 1997 (2), a structure of the PA pore has eluded researchers because of aggregation difficulties.In a prior study we used the chaperonin GroEL as a molecular scaffold to avoid this aggregation problem. We showed that the PA pore formed stable complexes with GroEL and solved a structure of a functional PA pore at ∼23-25 Å resolution by using negativestain EM (3). To gain a better structural understanding of this protein translocation machinery, it is imperative that one obtain structures of the pore inserted into membrane bilayers, and we chose a model bilayer system, the apolipoprotein A1-derived lipid nanodisc, for further studies (4). Unlike other systems such as liposomes or detergent micelles, nan...

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ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA + superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP␥S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP␥S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA + proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA + ATPases detected both six-and sevenfold particle symmetry.

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