The human sensor of double-stranded RNA (dsRNA) oligoadenylate synthetase 1 (hOAS1) polymerizes ATP into 2′,5′-linked iso-RNA (2-5A) involved in innate immunity, cell cycle, and differentiation. We report the crystal structure of hOAS1 in complex with dsRNA and 2′-deoxy ATP at 2.7 Å resolution, which reveals the mechanism of cytoplasmic dsRNA recognition and activation of oligoadenylate synthetases. Human OAS1 recognizes dsRNA using a previously uncharacterized protein/RNA interface that forms via a conformational change induced by binding of dsRNA. The protein/RNA interface involves two minor grooves and has no sequence-specific contacts, with the exception of a single hydrogen bond between the -NH 2 group of nucleobase G17 and the carbonyl oxygen of serine 56. Using a biochemical readout, we show that hOAS1 undergoes more than 20,000-fold activation upon dsRNA binding and that canonical or GU-wobble substitutions produce dsRNA mutants that retain either full or partial activity, in agreement with the crystal structure. Ultimately, the binding of dsRNA promotes an elaborate conformational rearrangement in the N-terminal lobe of hOAS1, which brings residues D75, D77, and D148 into proximity and creates coordination geometry for binding of two catalytic Mg 2+ ions and ATP. The assembly of this critical active-site structure provides the gate that couples binding of dsRNA to the production and downstream functions of 2-5A.ouble-stranded RNA (dsRNA)-binding oligoadenylate synthetases OAS1, OAS2, OAS3, OASL, and their splicing isoforms comprise the cohort of 10 homologous proteins either known to or implicated in 2′,5′-linked iso-RNA (2-5A) synthesis in human cells (1, 2). For OASL, the 2-5A synthesis activity has not been demonstrated and presently it is classified as catalytically inactive. Cells respond to 2-5A by activating the transcription factors IRF-3 and NF-κB and by mounting the IFN response (3-5). The 2-5A pathway serves as a conserved mammalian signal of viral presence providing resistance to hepatitis C virus (6), West Nile virus (7), and other RNA and DNA viruses (1,5,7,8). Broader roles of the 2-5A pathway in terminal differentiation of adipocytes (9), cell cycle (10), and BRCA1/IFN-γ-mediated apoptosis (11, 12) have emerged recently.Members of the OAS family belong to the nucleotidyl transferase superfamily that also includes poly-A polymerase (PAP1) (13) and CCA-adding enzyme (14). OAS1/2/3, PAP1, and CCA-adding enzyme synthesize RNA without using an oligonucleotide template. However, OAS1/2/3 have important distinctions: OAS1/2/3 synthesize 2′,5′-linked instead of 3′,5′-linked RNA; OAS1/2/3 do not require a prebound RNA primer; and, in contrast to the constitutively active PAP1 and CCA-adding enzyme, OAS1/2/3 are normally repressed and require binding of dsRNA for activity. The requirement for dsRNA binding reflects the unique biology of OAS1/2/3 as sensors of double-stranded RNA in the cytosol. It is largely unknown how the OAS family members recognize dsRNA and recruit it for regulation of 2-5A s...

Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant L-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.The ability to form spores is a remarkable property shared by many Bacillus species (23). Spores of these bacteria are formed in sporulation, a process triggered by starvation for one or more nutrients. These spores are metabolically dormant and extremely resistant to all manner of environmental stress factors (22, 23) and can remain in this dormant, resistant state for years. However, when conditions are favorable for growth, spores can rapidly return to life in the process of germination followed by outgrowth (11,19,21). A major signal that promotes germination is the presence of specific nutrients called germinants in the spore's environment. These nutrients are sensed by germinant receptors (GRs) located in the spore's inner membrane, and it is very likely that the binding of a nutrient germinant with its cognate GR triggers subsequent germination events.Bacillus subtilis spores contain three major GRs, termed GerA, GerB, and GerK (22, 23). The GerA GR responds to L-alanine or L-valine, while the GerB and GerK GRs cooperate to respond to a mixture of L-asparagine, D-glucose, D-fructose, and K ϩ ions (termed AGFK). Each of these GRs is encoded by a tricistronic operon, and the three polypeptides encoded by each operon (termed A, B, and C) are essential for specific GR activities and most likely form a multisubunit complex. The A and B subunits of each GR are likely to be polytopic integral membrane proteins, while the C subunit is a peripheral membrane protein held in the membrane by a diacy...