ABSTRACT
The Sm and Sm-like proteins are widely distributed among bacteria, archaea and eukarya. They participate in many processes related to RNA-processing and regulation of gene expression. While the function of the bacterial Lsm protein Hfq and eukaryotic Sm/Lsm proteins is rather well studied, the role of Lsm proteins in Archaea is investigated poorly. In this work, the RNA-binding ability of an archaeal Hfq-like protein from Methanococcus jannaschii has been studied by X-ray crystallography, anisotropy fluorescence and surface plasmon resonance. It has been found that MjaHfq preserves the proximal RNA-binding site that usually recognizes uridine-rich sequences. Distal adenine-binding and lateral RNA-binding sites show considerable structural changes as compared to bacterial Hfq. MjaHfq did not bind mononucleotides at these sites and would not recognize single-stranded RNA as its bacterial homologues. Nevertheless, MjaHfq possesses affinity to poly(A) RNA that seems to bind at the unstructured positive-charged N-terminal tail of the protein.
Subject(s)
Archaeal Proteins/chemistry , Host Factor 1 Protein/chemistry , Methanocaldococcus/chemistry , RNA, Archaeal/chemistry , RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Host Factor 1 Protein/genetics , Host Factor 1 Protein/metabolism , Kinetics , Methanocaldococcus/metabolism , Models, Molecular , Poly A/chemistry , Poly A/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In the presented paper, theoretical as well as electron microscopy and X-ray diffraction experimental approaches were employed for studding the process of Aß amyloid formation. Using quantitative estimates of a number of monomers which form the nuclei of amyloid fibrils the sizes of folding nuclei of amyloid fibrils for Aß40 and 42 have been determined for the first time. We have shown that the size of the primary nucleus of Aß42 peptide fibrils corresponds to 3 monomers, the size of the secondary nucleus for this peptide is 2 monomers. Applying the same analysis to Aß40 we conclude that the size of the primary nucleus is 2 monomers, and the size of the secondary nucleus is one monomer. Summation of our theoretical and experimental results has allowed us to propose a new model of the structural organization of amyloid fibrils. Our model suggests that the generation of fibrils takes place along the following simplified pathway: a monomerâa ring oligomerâa mature fibril consisting of ring oligomers. These data shed more light upon our understanding of what sizes of the oligomers could represent main targets for future therapies (tetramers for Aß42 and trimers for Aß40), and aid in the development of inhibitors of Aß40 and 42 oligomer formation.
