Limits...
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.

Show MeSH

Related in: MedlinePlus

Electrophoretic mobility shift assays of LGP2 RD and mutants with dsRNA. (A) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with 400 nM wild-type (wt) LGP2 RD or indicated RD mutants, respectively. (B) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with increasing concentrations (0, 0.4, 0.8, 1.6, 3.2 and 6.4 µM) of wt LGP2 RD and mutants P606→K, W604→A and H576→Y. For the wild-type RD two distinct bands appear shifted compared to free dsRNA at low protein concentrations already. The mutants show unspecific shifting and lower affinity to the RNA, indicated by remaining free dsRNA bands for all protein concentrations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665237&req=5

Figure 4: Electrophoretic mobility shift assays of LGP2 RD and mutants with dsRNA. (A) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with 400 nM wild-type (wt) LGP2 RD or indicated RD mutants, respectively. (B) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with increasing concentrations (0, 0.4, 0.8, 1.6, 3.2 and 6.4 µM) of wt LGP2 RD and mutants P606→K, W604→A and H576→Y. For the wild-type RD two distinct bands appear shifted compared to free dsRNA at low protein concentrations already. The mutants show unspecific shifting and lower affinity to the RNA, indicated by remaining free dsRNA bands for all protein concentrations.

Mentions: To test this idea further, we performed EMSAs with wild-type and mutant LGP2 RDs on dsRNA (Figure 4A and B). In general, the EMSAs confirm the results of the anisotropy measurements with respect to altered or retained dsRNA binding of site directed mutants (Figure 4A). Concentration-dependent analysis indicates that wild-type RD indeed shifts dsRNA to defined, specific bands. An initial shifted species with higher mobility is subsequently converted into a species with lower mobility (Figure 4B). These data suggest that LGP2 RD has two binding sites on the dsRNA ligand. One possibility is that RD specifically forms dimers on a single site on the dsRNA substrate. Since RD binds to dsRNA better than a hairpin of the same concentration, another explanation is that perhaps the RNA end structures contribute to binding and the two EMSA species are corresponding complexes with one and two RNA ends bound by protein. Dynamic light scattering, SAXS and analytical gel filtration suggest that RD is a monomer in the absence of RNA, and we do not see a convincing protein–protein dimer interface in the crystal lattice. In the presence of RNA our anisotropy studies, EMSAs as well as analytical gel filtration experiments can neither entirely eliminate nor verify the possibility of dimer formation. This is due to the resemblance in size between the RNA species and RD. Furthermore, judging from the dimension of the RD, more than one molecule could possibly bind to the 18–25 bp RNA duplexes used (Supplementary Table 2), independently of protein–protein interaction. Thus, analysis of an RNA-induced protein dimer formation must await further studies. In any case, we observe very defined species in the EMSAs, indicating a very specific interaction of LGP2 RD with the dsRNA substrate.Figure 4.


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)

Electrophoretic mobility shift assays of LGP2 RD and mutants with dsRNA. (A) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with 400 nM wild-type (wt) LGP2 RD or indicated RD mutants, respectively. (B) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with increasing concentrations (0, 0.4, 0.8, 1.6, 3.2 and 6.4 µM) of wt LGP2 RD and mutants P606→K, W604→A and H576→Y. For the wild-type RD two distinct bands appear shifted compared to free dsRNA at low protein concentrations already. The mutants show unspecific shifting and lower affinity to the RNA, indicated by remaining free dsRNA bands for all protein concentrations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Electrophoretic mobility shift assays of LGP2 RD and mutants with dsRNA. (A) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with 400 nM wild-type (wt) LGP2 RD or indicated RD mutants, respectively. (B) Retardation of 5′-Alexa Fluor 488-labeled dsRNA (25 bp, 60 nM) in a 10% native polyacrylamide gel after incubation with increasing concentrations (0, 0.4, 0.8, 1.6, 3.2 and 6.4 µM) of wt LGP2 RD and mutants P606→K, W604→A and H576→Y. For the wild-type RD two distinct bands appear shifted compared to free dsRNA at low protein concentrations already. The mutants show unspecific shifting and lower affinity to the RNA, indicated by remaining free dsRNA bands for all protein concentrations.
Mentions: To test this idea further, we performed EMSAs with wild-type and mutant LGP2 RDs on dsRNA (Figure 4A and B). In general, the EMSAs confirm the results of the anisotropy measurements with respect to altered or retained dsRNA binding of site directed mutants (Figure 4A). Concentration-dependent analysis indicates that wild-type RD indeed shifts dsRNA to defined, specific bands. An initial shifted species with higher mobility is subsequently converted into a species with lower mobility (Figure 4B). These data suggest that LGP2 RD has two binding sites on the dsRNA ligand. One possibility is that RD specifically forms dimers on a single site on the dsRNA substrate. Since RD binds to dsRNA better than a hairpin of the same concentration, another explanation is that perhaps the RNA end structures contribute to binding and the two EMSA species are corresponding complexes with one and two RNA ends bound by protein. Dynamic light scattering, SAXS and analytical gel filtration suggest that RD is a monomer in the absence of RNA, and we do not see a convincing protein–protein dimer interface in the crystal lattice. In the presence of RNA our anisotropy studies, EMSAs as well as analytical gel filtration experiments can neither entirely eliminate nor verify the possibility of dimer formation. This is due to the resemblance in size between the RNA species and RD. Furthermore, judging from the dimension of the RD, more than one molecule could possibly bind to the 18–25 bp RNA duplexes used (Supplementary Table 2), independently of protein–protein interaction. Thus, analysis of an RNA-induced protein dimer formation must await further studies. In any case, we observe very defined species in the EMSAs, indicating a very specific interaction of LGP2 RD with the dsRNA substrate.Figure 4.

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