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The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex. However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to ∼ 100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres.

The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.

The limited proteolysis of the Bacillus stearothermophilus pyruvate dehydrogenase complex by V8 protease yields its core structure solely composed of lipoate acetyltransferase (E2) fragments. The changes in the core with guanidine hydrochloride (GdnHCl) were biphasic: below 0.8 M (first) and above 1.0 M (second) GdnHCl. The changes in the first phase were slight but significant: decreases in ellipticity and light scattering, and an increase in E2 activity. Insignificant changes in the molecular shape and size of the core were detected on fluorescence spectroscopy, ultracentrifugation, gel filtration, and electron microscopy. On the other hand, the changes in the second phase were drastic; the core was disassembled and denatured.