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The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA.

Pippig DA, Hellmuth JC, Cui S, Kirchhofer A, Lammens K, Lammens A, Schmidt A, Rothenfusser S, Hopfner KP - Nucleic Acids Res. (2009)

Bottom Line: We identify conserved and receptor-specific parts of the RNA binding site.Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors.Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Gene Center, Ludwig-Maximilians University Munich, Munich, Germany.

ABSTRACT
RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-I-like receptor, LGP2, differentially regulates RIG-I- and MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5'-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6 A crystal structure of LGP2 RD along with in vitro and in vivo functional analyses and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5'-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

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Binding of dsRNA to LGP2 RD. (A) Fluorescence anisotropy changes of a 5′-Alexa Fluor 488-labeled 25 bp RNA duplex (37 nM) in response to titration with wild-type LGP2 RD (filled square, Kd=68 ± 6 nM) and various mutants, respectively. Control mutation N583→D (open diamond, Kd=38 ± 4 nM) located on the convex site of RD and mutation K626→E (open left-facing triangle, Kd=51 ± 5 nM) do not show significantly altered dsRNA binding affinity. Affinities for mutants L621→A (filled up-facing triangle, Kd=165 ± 26 nM) and K605→E (filled down-facing triangle, Kd=140 ± 16 nM) are slightly decreased, while a mutation of K634→E (filled circle) completely suppresses binding. A decrease of binding affinity, but increase in maximum reached anisotropy signal is seen for P606→K (open right-facing triangle, Kd=230 ± 10 nM), W604→A (open square, Kd=136 ± 6 nM) and H576→Y (open circle, Kd=304 ± 10 nM). (B) Competition of binding of an Alexa Fluor 488-5-U-labeled hairpin RNA (in vitro transcriped, 40 nM) to LGP2 RD (470 nM) by stepwise addition of different non-fluorescent RNA species (synthetic 5′OH/5′OH dsRNA: 19 bp; 5′PPP/5′OH dsRNA: 19 bp; 5′OH ssRNA: 19n; 5′PPP ssRNA: 19n; and in vitro transcribed 5′PPP hairpin: 18 bp ± 4n) followed by fluorescence anisotropy. Data points were connected for better outline.
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Figure 3: Binding of dsRNA to LGP2 RD. (A) Fluorescence anisotropy changes of a 5′-Alexa Fluor 488-labeled 25 bp RNA duplex (37 nM) in response to titration with wild-type LGP2 RD (filled square, Kd=68 ± 6 nM) and various mutants, respectively. Control mutation N583→D (open diamond, Kd=38 ± 4 nM) located on the convex site of RD and mutation K626→E (open left-facing triangle, Kd=51 ± 5 nM) do not show significantly altered dsRNA binding affinity. Affinities for mutants L621→A (filled up-facing triangle, Kd=165 ± 26 nM) and K605→E (filled down-facing triangle, Kd=140 ± 16 nM) are slightly decreased, while a mutation of K634→E (filled circle) completely suppresses binding. A decrease of binding affinity, but increase in maximum reached anisotropy signal is seen for P606→K (open right-facing triangle, Kd=230 ± 10 nM), W604→A (open square, Kd=136 ± 6 nM) and H576→Y (open circle, Kd=304 ± 10 nM). (B) Competition of binding of an Alexa Fluor 488-5-U-labeled hairpin RNA (in vitro transcriped, 40 nM) to LGP2 RD (470 nM) by stepwise addition of different non-fluorescent RNA species (synthetic 5′OH/5′OH dsRNA: 19 bp; 5′PPP/5′OH dsRNA: 19 bp; 5′OH ssRNA: 19n; 5′PPP ssRNA: 19n; and in vitro transcribed 5′PPP hairpin: 18 bp ± 4n) followed by fluorescence anisotropy. Data points were connected for better outline.

Mentions: To learn more about the functional sites of LGP2 RD, we first analyzed whether it can directly bind RNA and what RNA structures or epitopes might contribute to binding. The physiological ligand for LGP2 is unknown. Its negative regulation of RIG-I-dependent signaling in vivo may result from competition for viral RNA and thus could be directed against two RIG-I PAMPs, 5′-triphosphate RNA and dsRNA. Since full-length LGP2 has been shown to bind to dsRNA with a higher preference than ssRNA, we hypothesized that RD of LGP2 might be one of its dsRNA recognizing elements. We tested several RNA oligonucleotides (Supplementary Table 2) in equilibrium binding experiments using fluorescence polarization anisotropy measurements. Indeed, we find that LGP2 RD binds to a dsRNA 25-mer with quite high affinity. We can fit the binding isotherm with a single site-binding model and using this model, obtain an apparent Kd of 68 ± 6 nM (Figure 3A).Figure 3.


The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA.

Pippig DA, Hellmuth JC, Cui S, Kirchhofer A, Lammens K, Lammens A, Schmidt A, Rothenfusser S, Hopfner KP - Nucleic Acids Res. (2009)

Binding of dsRNA to LGP2 RD. (A) Fluorescence anisotropy changes of a 5′-Alexa Fluor 488-labeled 25 bp RNA duplex (37 nM) in response to titration with wild-type LGP2 RD (filled square, Kd=68 ± 6 nM) and various mutants, respectively. Control mutation N583→D (open diamond, Kd=38 ± 4 nM) located on the convex site of RD and mutation K626→E (open left-facing triangle, Kd=51 ± 5 nM) do not show significantly altered dsRNA binding affinity. Affinities for mutants L621→A (filled up-facing triangle, Kd=165 ± 26 nM) and K605→E (filled down-facing triangle, Kd=140 ± 16 nM) are slightly decreased, while a mutation of K634→E (filled circle) completely suppresses binding. A decrease of binding affinity, but increase in maximum reached anisotropy signal is seen for P606→K (open right-facing triangle, Kd=230 ± 10 nM), W604→A (open square, Kd=136 ± 6 nM) and H576→Y (open circle, Kd=304 ± 10 nM). (B) Competition of binding of an Alexa Fluor 488-5-U-labeled hairpin RNA (in vitro transcriped, 40 nM) to LGP2 RD (470 nM) by stepwise addition of different non-fluorescent RNA species (synthetic 5′OH/5′OH dsRNA: 19 bp; 5′PPP/5′OH dsRNA: 19 bp; 5′OH ssRNA: 19n; 5′PPP ssRNA: 19n; and in vitro transcribed 5′PPP hairpin: 18 bp ± 4n) followed by fluorescence anisotropy. Data points were connected for better outline.
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Figure 3: Binding of dsRNA to LGP2 RD. (A) Fluorescence anisotropy changes of a 5′-Alexa Fluor 488-labeled 25 bp RNA duplex (37 nM) in response to titration with wild-type LGP2 RD (filled square, Kd=68 ± 6 nM) and various mutants, respectively. Control mutation N583→D (open diamond, Kd=38 ± 4 nM) located on the convex site of RD and mutation K626→E (open left-facing triangle, Kd=51 ± 5 nM) do not show significantly altered dsRNA binding affinity. Affinities for mutants L621→A (filled up-facing triangle, Kd=165 ± 26 nM) and K605→E (filled down-facing triangle, Kd=140 ± 16 nM) are slightly decreased, while a mutation of K634→E (filled circle) completely suppresses binding. A decrease of binding affinity, but increase in maximum reached anisotropy signal is seen for P606→K (open right-facing triangle, Kd=230 ± 10 nM), W604→A (open square, Kd=136 ± 6 nM) and H576→Y (open circle, Kd=304 ± 10 nM). (B) Competition of binding of an Alexa Fluor 488-5-U-labeled hairpin RNA (in vitro transcriped, 40 nM) to LGP2 RD (470 nM) by stepwise addition of different non-fluorescent RNA species (synthetic 5′OH/5′OH dsRNA: 19 bp; 5′PPP/5′OH dsRNA: 19 bp; 5′OH ssRNA: 19n; 5′PPP ssRNA: 19n; and in vitro transcribed 5′PPP hairpin: 18 bp ± 4n) followed by fluorescence anisotropy. Data points were connected for better outline.
Mentions: To learn more about the functional sites of LGP2 RD, we first analyzed whether it can directly bind RNA and what RNA structures or epitopes might contribute to binding. The physiological ligand for LGP2 is unknown. Its negative regulation of RIG-I-dependent signaling in vivo may result from competition for viral RNA and thus could be directed against two RIG-I PAMPs, 5′-triphosphate RNA and dsRNA. Since full-length LGP2 has been shown to bind to dsRNA with a higher preference than ssRNA, we hypothesized that RD of LGP2 might be one of its dsRNA recognizing elements. We tested several RNA oligonucleotides (Supplementary Table 2) in equilibrium binding experiments using fluorescence polarization anisotropy measurements. Indeed, we find that LGP2 RD binds to a dsRNA 25-mer with quite high affinity. We can fit the binding isotherm with a single site-binding model and using this model, obtain an apparent Kd of 68 ± 6 nM (Figure 3A).Figure 3.

Bottom Line: We identify conserved and receptor-specific parts of the RNA binding site.Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors.Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Gene Center, Ludwig-Maximilians University Munich, Munich, Germany.

ABSTRACT
RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-I-like receptor, LGP2, differentially regulates RIG-I- and MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5'-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6 A crystal structure of LGP2 RD along with in vitro and in vivo functional analyses and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5'-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

Show MeSH
Related in: MedlinePlus