Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1

Abstract

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.

Fig. 1.

Structure of the hOAS1●dsRNA●dATP ternary complex. The complex is shown in two orientations related by 90° rotation. Protein is shown as ribbons; dsRNA (purple) and dATP (light blue) are shown as molecular surfaces. Two Mg²⁺ ions are shown as spheres (green). dsRNA is bound at the junction of the N lobe and the C lobe of hOAS1.

Fig. 2.

Protein/RNA interface and the 2-5A synthesis activity of hOAS1. (A) Overview of the protein/RNA interface formed upon recognition of two dsRNA minor grooves by hOAS1. The minor groove with base pairs 13–18 is recognized by the protein residues located predominantly in the N lobe of hOAS1. The minor groove with base pairs 2–8 is recognized by the protein residues located predominantly in the C lobe of hOAS1. (B) Ten-minute end point assay for hOAS1 activation by dsRNA. Synthesis of a range of 2-5A species was analyzed by gel electrophoresis using α³²p-ATP for visualization (Methods). (C) Rate constants (k_obs) for 2-5A synthesis obtained with wild-type and mutant dsRNA using single-exponential fitting of time courses. Error bars show SEs from two measurements of k_obs.

Fig. 3.

Global conformational changes in OAS1 upon dsRNA binding. (A) Side view of the interlobe groove in pOAS1 and in hOAS1. Clipping planes cut the structures at the same position. (B) Superposition of pOAS1 in the inactive conformation (PDB code 1PX5) with hOAS1 in the active conformation (present work). Movement of helix N5 away from the protein/RNA interface and sliding of the β-strand floor that contains the active site residues D75, D77, and D148 are shown.

Fig. 4.

RNA-induced conformational changes in the active site. (A) Binding of dsRNA extracts arginine R195 from the core of the protein and positions it at the protein/RNA interface. The exchange between residues K66 and R195 is coupled to the formation of the new helix, N4. The movement of the amino acids is shown by red arrows. (B) Conformational rearrangement positions the active site residues D75, D77, and D148 compactly for coordination of two Mg²⁺ ions (green spheres) and for binding of dATP. Red triangles show the inactive and active arrangements of the acidic triad.

Fig. 5.

Model for OAS1 regulation by dsRNA. (A) Comparison of the helix N4 in OAS1 (Left) with the structurally equivalent helix in poly-A polymerase PAP1 (Right; PDB ID 2Q66). (B) Active site residues of OAS1 D75, D77, and D148 face divergent directions in the ground state and are not compatible with coordination of Mg²⁺ ions and binding of ATP. dsRNA induces RICS and brings the residues D75, D77, and D148 together to enable binding of ATP●Mg²⁺. It is possible that ATP stabilizes RICS further upon binding to the OAS1●dsRNA complex, finalizing the conformational change. The dsRNA-independent CCA-adding enzyme and PAP1 are locked in the constitutively active state because their RICS-equivalent structures contain bulky hydrophobic residues anchored in the protein core.