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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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Comparison of RDs of RIG-I-like helicases. (A) Small angle X-ray scattering curves of RIG-I RD and MDA5 RD (upper panel, merged from curves obtained with protein concentrations of 2, 5 and 10 mg/ml). The lower panel shows the pair distribution functions for RIG-I RD and MDA5 RD derived from raw data with Gnome (ATSAS 2.1). (B) Superimposition of SAXS surface models of RIG-I (left) and MDA5 RD (right) with crystal structures of RIG-I RD (yellow, brown) and a model of MDA5 RD (model based on RIG-I and LGP2 RD crystal structure, blue, light blue), respectively. (C) Electrostatic surface potential (ranging from blue=9 kT/e to red=−9 kT/e) of LGP2 RD, RIG-I RD (from crystal structure) and a model of MDA5 RD. Residues crucial for general RNA interaction and either 5′-triphosphate RNA specificity in RIG-I (K888, buried K858) or dsRNA specificity in LGP2 (corresponding K634, P606 and additionally buried W604 and K605) are highlighted as well as corresponding residues for MDA5 RD (K983, buried I956).
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Figure 5: Comparison of RDs of RIG-I-like helicases. (A) Small angle X-ray scattering curves of RIG-I RD and MDA5 RD (upper panel, merged from curves obtained with protein concentrations of 2, 5 and 10 mg/ml). The lower panel shows the pair distribution functions for RIG-I RD and MDA5 RD derived from raw data with Gnome (ATSAS 2.1). (B) Superimposition of SAXS surface models of RIG-I (left) and MDA5 RD (right) with crystal structures of RIG-I RD (yellow, brown) and a model of MDA5 RD (model based on RIG-I and LGP2 RD crystal structure, blue, light blue), respectively. (C) Electrostatic surface potential (ranging from blue=9 kT/e to red=−9 kT/e) of LGP2 RD, RIG-I RD (from crystal structure) and a model of MDA5 RD. Residues crucial for general RNA interaction and either 5′-triphosphate RNA specificity in RIG-I (K888, buried K858) or dsRNA specificity in LGP2 (corresponding K634, P606 and additionally buried W604 and K605) are highlighted as well as corresponding residues for MDA5 RD (K983, buried I956).

Mentions: We have not been able to crystallize the RD of MDA5, so we used small angle X-ray scattering as well as homology modeling to generate a first structural draft of MDA5 RD (Figure 5). Our construct of MDA5 RD spans residues 897–1025, similar to the used LGP2 and RIG-I RDs. Small angle X-ray scattering data were collected at DESY beamline X33 and data processed with programs from the ATSAS 2.1 suite (43) (Figure 5A). We noticed some limited aggregation in RIG-I and MDA5 RD samples at higher concentrations, so very low-resolution data needed to be truncated. Using GASBORp, we generated ab initio reconstructions using the dummy residue approach (Figure 5B). In the case of RIG-I RD, the solution scattering data correlate well with size and shape of the respective crystal structures. Likewise, an ab initio dummy residue model matches the shape and size of the crystals structure to a high degree.Figure 5.


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)

Comparison of RDs of RIG-I-like helicases. (A) Small angle X-ray scattering curves of RIG-I RD and MDA5 RD (upper panel, merged from curves obtained with protein concentrations of 2, 5 and 10 mg/ml). The lower panel shows the pair distribution functions for RIG-I RD and MDA5 RD derived from raw data with Gnome (ATSAS 2.1). (B) Superimposition of SAXS surface models of RIG-I (left) and MDA5 RD (right) with crystal structures of RIG-I RD (yellow, brown) and a model of MDA5 RD (model based on RIG-I and LGP2 RD crystal structure, blue, light blue), respectively. (C) Electrostatic surface potential (ranging from blue=9 kT/e to red=−9 kT/e) of LGP2 RD, RIG-I RD (from crystal structure) and a model of MDA5 RD. Residues crucial for general RNA interaction and either 5′-triphosphate RNA specificity in RIG-I (K888, buried K858) or dsRNA specificity in LGP2 (corresponding K634, P606 and additionally buried W604 and K605) are highlighted as well as corresponding residues for MDA5 RD (K983, buried I956).
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Figure 5: Comparison of RDs of RIG-I-like helicases. (A) Small angle X-ray scattering curves of RIG-I RD and MDA5 RD (upper panel, merged from curves obtained with protein concentrations of 2, 5 and 10 mg/ml). The lower panel shows the pair distribution functions for RIG-I RD and MDA5 RD derived from raw data with Gnome (ATSAS 2.1). (B) Superimposition of SAXS surface models of RIG-I (left) and MDA5 RD (right) with crystal structures of RIG-I RD (yellow, brown) and a model of MDA5 RD (model based on RIG-I and LGP2 RD crystal structure, blue, light blue), respectively. (C) Electrostatic surface potential (ranging from blue=9 kT/e to red=−9 kT/e) of LGP2 RD, RIG-I RD (from crystal structure) and a model of MDA5 RD. Residues crucial for general RNA interaction and either 5′-triphosphate RNA specificity in RIG-I (K888, buried K858) or dsRNA specificity in LGP2 (corresponding K634, P606 and additionally buried W604 and K605) are highlighted as well as corresponding residues for MDA5 RD (K983, buried I956).
Mentions: We have not been able to crystallize the RD of MDA5, so we used small angle X-ray scattering as well as homology modeling to generate a first structural draft of MDA5 RD (Figure 5). Our construct of MDA5 RD spans residues 897–1025, similar to the used LGP2 and RIG-I RDs. Small angle X-ray scattering data were collected at DESY beamline X33 and data processed with programs from the ATSAS 2.1 suite (43) (Figure 5A). We noticed some limited aggregation in RIG-I and MDA5 RD samples at higher concentrations, so very low-resolution data needed to be truncated. Using GASBORp, we generated ab initio reconstructions using the dummy residue approach (Figure 5B). In the case of RIG-I RD, the solution scattering data correlate well with size and shape of the respective crystal structures. Likewise, an ab initio dummy residue model matches the shape and size of the crystals structure to a high degree.Figure 5.

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