Molecular insights into intra-complex signal transmission during stressosome activation.

The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin.

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (KD values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc)n oligomers (n = 1-6). KD values were found to range between approximately 10-35 microM. The strength of the interactions between the C fragment and (NeuAc)n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells.