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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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Structure guided site directed mutagenesis of LGP2 RD. (A) Structure-based sequence alignment of the C-terminal regions of human LGP2, RIG-I and MDA5. Secondary structure elements corresponding to LGP2 RD are shown above the alignment. Invariant residues are highlighted with red background, conserved ones in red font. The zinc-coordinating cysteines are marked with asterisks. (B) Localization of mutations, shown in a cartoon representation with electrostatic surface potential (ranging from blue=5 kT/e to red=−5 kT/e). The model is shown in ‘standard view’, used in all other figures (left) and 180° rotated around the vertical axis. Mutated residues are highlighted with sticks (cyan).
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Figure 2: Structure guided site directed mutagenesis of LGP2 RD. (A) Structure-based sequence alignment of the C-terminal regions of human LGP2, RIG-I and MDA5. Secondary structure elements corresponding to LGP2 RD are shown above the alignment. Invariant residues are highlighted with red background, conserved ones in red font. The zinc-coordinating cysteines are marked with asterisks. (B) Localization of mutations, shown in a cartoon representation with electrostatic surface potential (ranging from blue=5 kT/e to red=−5 kT/e). The model is shown in ‘standard view’, used in all other figures (left) and 180° rotated around the vertical axis. Mutated residues are highlighted with sticks (cyan).

Mentions: With the exception of some differences in loop regions that connect secondary structure, the fold of LGP2 RD is highly related to the fold of RIG-I RD (Figure 1C). The RMSD, as calculated by LSQMAN (44), between the Cα atoms of LGP2 (molecule C) and RIG-I RD is 1.178 Å for 108 matched residues (Supplementary Figure 1B). In particular, the backbone geometry of the metal coordinating cluster is, with the exception of one loop insertion in RIG-I, conserved between RIG-I and LGP2 (Figures 1 and 2A). The metal in LGP2 is coordinated in a tetrahedral manner by the sulfur atoms of the cysteine cluster C556, C559, C612 and C615. Although we have a mercury ion in our crystal form we think the geometry is similar in the presence of zinc, since crystal structures of RIG-I RD in the presence of mercury and zinc turned out to be virtually identical (27). W619LGP2—conserved between LGP2, RIG-I and MDA5—bounds this cluster towards the core of the domain. Thus, correct formation of the metal coordination sphere is presumably necessary for fold of RDs, which might explain the severe effect of point mutation in the zinc-binding cluster on activity of RIG-I (27). In support of this, point mutation in metal coordinating cysteine residues resulted in an unstable LGP2 RD, which could not be over-expressed in E. coli in our hands. A notable difference in the metal binding site between RIG-I and LGP2 RD is the loop, which connects C612LGP2 and C615LGP2 (Figure 1C). In RIG-I this loop is two residues longer, forming a short β-turn. In MDA5, this loop is one amino acid shorter indicating that this region could account for functional differences between RIG-I-like helicases (Figure 2A).Figure 2.


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)

Structure guided site directed mutagenesis of LGP2 RD. (A) Structure-based sequence alignment of the C-terminal regions of human LGP2, RIG-I and MDA5. Secondary structure elements corresponding to LGP2 RD are shown above the alignment. Invariant residues are highlighted with red background, conserved ones in red font. The zinc-coordinating cysteines are marked with asterisks. (B) Localization of mutations, shown in a cartoon representation with electrostatic surface potential (ranging from blue=5 kT/e to red=−5 kT/e). The model is shown in ‘standard view’, used in all other figures (left) and 180° rotated around the vertical axis. Mutated residues are highlighted with sticks (cyan).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665237&req=5

Figure 2: Structure guided site directed mutagenesis of LGP2 RD. (A) Structure-based sequence alignment of the C-terminal regions of human LGP2, RIG-I and MDA5. Secondary structure elements corresponding to LGP2 RD are shown above the alignment. Invariant residues are highlighted with red background, conserved ones in red font. The zinc-coordinating cysteines are marked with asterisks. (B) Localization of mutations, shown in a cartoon representation with electrostatic surface potential (ranging from blue=5 kT/e to red=−5 kT/e). The model is shown in ‘standard view’, used in all other figures (left) and 180° rotated around the vertical axis. Mutated residues are highlighted with sticks (cyan).
Mentions: With the exception of some differences in loop regions that connect secondary structure, the fold of LGP2 RD is highly related to the fold of RIG-I RD (Figure 1C). The RMSD, as calculated by LSQMAN (44), between the Cα atoms of LGP2 (molecule C) and RIG-I RD is 1.178 Å for 108 matched residues (Supplementary Figure 1B). In particular, the backbone geometry of the metal coordinating cluster is, with the exception of one loop insertion in RIG-I, conserved between RIG-I and LGP2 (Figures 1 and 2A). The metal in LGP2 is coordinated in a tetrahedral manner by the sulfur atoms of the cysteine cluster C556, C559, C612 and C615. Although we have a mercury ion in our crystal form we think the geometry is similar in the presence of zinc, since crystal structures of RIG-I RD in the presence of mercury and zinc turned out to be virtually identical (27). W619LGP2—conserved between LGP2, RIG-I and MDA5—bounds this cluster towards the core of the domain. Thus, correct formation of the metal coordination sphere is presumably necessary for fold of RDs, which might explain the severe effect of point mutation in the zinc-binding cluster on activity of RIG-I (27). In support of this, point mutation in metal coordinating cysteine residues resulted in an unstable LGP2 RD, which could not be over-expressed in E. coli in our hands. A notable difference in the metal binding site between RIG-I and LGP2 RD is the loop, which connects C612LGP2 and C615LGP2 (Figure 1C). In RIG-I this loop is two residues longer, forming a short β-turn. In MDA5, this loop is one amino acid shorter indicating that this region could account for functional differences between RIG-I-like helicases (Figure 2A).Figure 2.

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