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Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate.

Lu C, Ranjith-Kumar CT, Hao L, Kao CC, Li P - Nucleic Acids Res. (2010)

Bottom Line: Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA.Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I.These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.

ABSTRACT
RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

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Mutations at the blunt-ended dsRNA-binding surface affect RNA binding by RIG-I CTD. (A) Binding studies of wild-type and mutants of RIG-I CTD with the 14-bp blunt-ended dsRNA by EMSA. (B) Binding studies of RIG-I CTD mutants with a 14-bp 5′ ppp dsRNA. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.
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Figure 4: Mutations at the blunt-ended dsRNA-binding surface affect RNA binding by RIG-I CTD. (A) Binding studies of wild-type and mutants of RIG-I CTD with the 14-bp blunt-ended dsRNA by EMSA. (B) Binding studies of RIG-I CTD mutants with a 14-bp 5′ ppp dsRNA. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.

Mentions: The structure of RIG-I CTD bound to the blunt-ended dsRNA indicates that residues Arg811, Lys812 and His871 may play critical roles in the specific binding of blunt-ended dsRNA. To test this prediction, we purified RIG-I CTD mutants R811E, R811S, K812E, K812S, H871E and conducted RNA-binding studies (Figure 4 and Supplementary Figure S2). Substitution of Arg811 with glutamate almost abolished blunt-ended dsRNA binding and significantly reduced binding to both 5′-ppp dsRNA and ssRNA (Figure 4). Replacement of Arg811 by a hydrophilic serine reduced the binding of blunt-ended dsRNA, but did not affect binding to triphosphorylated dsRNA or ssRNA (Figure 4). Substitution of the nearby Lys812 by glutamate dramatically reduced blunt-ended dsRNA binding, but only slightly reduced binding to triphosphorylated dsRNA or ssRNA (Figure 4). Replacement of Lys812 by a neutral serine residue did not affect RNA binding (Supplementary Figure S3). Surprisingly, the replacement of His871 by glutamate did not affect the binding to all three forms of RNA (Figure 4), suggesting that the His871 makes only minimal contribution to RNA binding. As expected, the replacement of Arg866, a residue not at the RNA-binding surface, by glutamate did not affect RNA binding (Figure 4). The slightly slower migration of the R866E and H871E complex is likely due to the changes of the surface electrostatics of the mutant proteins. In addition, our previous studies showed that mutation K907E disrupted blunt-ended dsRNA as well as 5′-ppp dsRNA binding (30). Together, these results suggest that the electrostatic interactions between Arg811 and Lys907 and the dsRNA are essential for blunt-ended dsRNA binding. Furthermore, previous studies showed that substitution of Phe853 by serine also reduced dsRNA binding (30), suggesting the hydrophobic interaction between Phe853 and the exposed base pair is also needed for effective RNA binding.Figure 4.


Crystal structure of RIG-I C-terminal domain bound to blunt-ended double-strand RNA without 5' triphosphate.

Lu C, Ranjith-Kumar CT, Hao L, Kao CC, Li P - Nucleic Acids Res. (2010)

Mutations at the blunt-ended dsRNA-binding surface affect RNA binding by RIG-I CTD. (A) Binding studies of wild-type and mutants of RIG-I CTD with the 14-bp blunt-ended dsRNA by EMSA. (B) Binding studies of RIG-I CTD mutants with a 14-bp 5′ ppp dsRNA. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.
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Figure 4: Mutations at the blunt-ended dsRNA-binding surface affect RNA binding by RIG-I CTD. (A) Binding studies of wild-type and mutants of RIG-I CTD with the 14-bp blunt-ended dsRNA by EMSA. (B) Binding studies of RIG-I CTD mutants with a 14-bp 5′ ppp dsRNA. (C) Binding studies of RIG-I CTD mutants with a 13-nt 5′ ppp ssRNA.
Mentions: The structure of RIG-I CTD bound to the blunt-ended dsRNA indicates that residues Arg811, Lys812 and His871 may play critical roles in the specific binding of blunt-ended dsRNA. To test this prediction, we purified RIG-I CTD mutants R811E, R811S, K812E, K812S, H871E and conducted RNA-binding studies (Figure 4 and Supplementary Figure S2). Substitution of Arg811 with glutamate almost abolished blunt-ended dsRNA binding and significantly reduced binding to both 5′-ppp dsRNA and ssRNA (Figure 4). Replacement of Arg811 by a hydrophilic serine reduced the binding of blunt-ended dsRNA, but did not affect binding to triphosphorylated dsRNA or ssRNA (Figure 4). Substitution of the nearby Lys812 by glutamate dramatically reduced blunt-ended dsRNA binding, but only slightly reduced binding to triphosphorylated dsRNA or ssRNA (Figure 4). Replacement of Lys812 by a neutral serine residue did not affect RNA binding (Supplementary Figure S3). Surprisingly, the replacement of His871 by glutamate did not affect the binding to all three forms of RNA (Figure 4), suggesting that the His871 makes only minimal contribution to RNA binding. As expected, the replacement of Arg866, a residue not at the RNA-binding surface, by glutamate did not affect RNA binding (Figure 4). The slightly slower migration of the R866E and H871E complex is likely due to the changes of the surface electrostatics of the mutant proteins. In addition, our previous studies showed that mutation K907E disrupted blunt-ended dsRNA as well as 5′-ppp dsRNA binding (30). Together, these results suggest that the electrostatic interactions between Arg811 and Lys907 and the dsRNA are essential for blunt-ended dsRNA binding. Furthermore, previous studies showed that substitution of Phe853 by serine also reduced dsRNA binding (30), suggesting the hydrophobic interaction between Phe853 and the exposed base pair is also needed for effective RNA binding.Figure 4.

Bottom Line: Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA.Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I.These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.

ABSTRACT
RIG-I recognizes molecular patterns in viral RNA to regulate the induction of type I interferons. The C-terminal domain (CTD) of RIG-I exhibits high affinity for 5' triphosphate (ppp) dsRNA as well as blunt-ended dsRNA. Structures of RIG-I CTD bound to 5'-ppp dsRNA showed that RIG-I recognizes the termini of dsRNA and interacts with the ppp through electrostatic interactions. However, the structural basis for the recognition of non-phosphorylated dsRNA by RIG-I is not fully understood. Here, we show that RIG-I CTD binds blunt-ended dsRNA in a different orientation compared to 5' ppp dsRNA and interacts with both strands of the dsRNA. Overlapping sets of residues are involved in the recognition of blunt-ended dsRNA and 5' ppp dsRNA. Mutations at the RNA-binding surface affect RNA binding and signaling by RIG-I. These results provide the mechanistic basis for how RIG-I recognizes different RNA ligands.

Show MeSH