In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.The soil-dwelling, spore-forming B. cereus bacterium 1-3 produces a number of virulence factors 4 including several secreted pore-forming toxins (PFTs) that form lytic channels in the membranes of target cells 5 . One of these toxins, hemolysin II (HlyII), is present in several closely related Bacillus species including B. cereus, B. thuringiensis (a bacterium that parasitizes insects and has insecticide applications), and B. anthracis (the cause of anthrax) 6, 7 . In B. cereus, expression of HlyII is under the control of the HlyIIR protein 8 , and the Fur system that regulates iron homeostasis 9,10 . Expression of HlyII is greater under oxic conditions than under conditions mimicking the intestinal tract, suggesting the toxin may not play a major role in gastrointestinal disease 11 . Although the physiological target of HlyII is not known, the purified toxin lyses rabbit and human erythrocytes 12 as well as other cultured mammalian cells 13,14 . In addition, the toxin can attack species like algae 15 and insects. Studies in mice and insects suggest that HlyII is involved in virulence, and that the toxin causes apoptosis of macrophages in vitro and in vivo 16 . HlyII belongs to a larger family of secreted toxins with similar predicted core structures including the B. cereus cytotoxin K (CytK) 17 , Staphylococcal hemolysins/leukocidins 18,19 , and toxins secreted by a variety of Vibrio species 20 . Similar to other family members, HlyII is secreted as a water-soluble monomer that assembles into a heptameric pore following binding to cell membranes 12,21 . A unique feature of HlyII is the attachment of a C-terminal domain consisting of 94 amino acids that shows no sequence or structural homology to other known proteins 18 . The C-terminal domain, henceforth r...

Pathogenic bacteria secrete pore-forming toxins (PFTs) to selectively defend against immune cells and to break through cellular barriers in the host. Understanding how PFTs attack cell membranes is not only essential for therapeutic intervention but for designing agents to deliver drugs to specific human cell subtypes, for example in anti-cancer or anti-viral therapies. Many toxins contain accessory domains that help recognize specific molecular epitopes on the membranes of target cells, including proteins, carbohydrates, and lipids. Here we report NMR assignments for the 94-residue 10 kDa C-terminal accessory domain of Bacillus cereus hemolysin II, HlyIIC, that has no known structural or functional homologues. The HlyIIC domain exists in a dynamic equilibrium due to cis/trans isomerization of its Gly86-Pro87 peptide bond. The cis and trans forms are about equally populated and are in slow exchange on the NMR timescale, giving rise to separate signals for approximately half of the residues in the domain. Assignments for the cis and trans forms were achieved with the aid of a P87M mutant that stabilizes the trans form, and separate sequential walks for the two forms in 3D NMR spectra of the wild-type HlyIIC. Based on backbone chemical shifts, the domain has a α1-α2-β1-β2-β3-β4-α3-β5 order of secondary structure elements. The last strand in the trans form and in the P87M mutant is shortened near Pro87 compared to the cis form. Both cis/trans isomerization and the P87M mutation cause large chemical shift changes throughout HlyIIC, suggesting that the proline is important in stabilizing the structure of the domain. The NMR assignments pave the way for solving the structures of the multiple conformational forms of HlyIIC and establishing their mechanism of interconversion.

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of H-N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, C rotating frame T relaxation (T) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

The C‐terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans‐membrane‐pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M‐HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT‐HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain‐swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT‐HlyIIC strongly disfavors the dimer compared to the P405M‐HlyIIC or P405A‐HlyIIC mutants. The WT proline may thus act as a “gatekeeper,” warding off aggregative misfolding.