Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was carried out. These molecules differ only by a single amino acid at the floor of the peptide binding groove. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties.
Streptococcus pyogenes pSM19035-encoded epsilon (10.7 kDa) and zeta (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with zeta acting as toxin that kills plasmid-deprived cells and epsilon as an antitoxin that neutralises the activity of zeta. The epsilon and zeta proteins co-purify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as epsilon2zeta2 heterotetramer in solution. Co-crystals of the epsilon2zeta2 complex contain epsilon and zeta in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the zeta protein has a significantly lower thermodynamic stability than the epsilon protein both in free state and in the complex. Proteolytic studies indicate that zeta protein is more stable in the epsilon2zeta2 complex than in the free state. In vivo studies reveal a short half-life of the epsilon antitoxin (-18 min) and a long lifetime of the zeta toxin (>60 min). When transcription-translation of a plasmid containing the epsilon and zeta genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the epsilon protein from the background.
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