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Assembly-driven activation of the AIM2 foreign-dsDNA sensor provides a polymerization template for downstream ASC.

Morrone SR, Matyszewski M, Yu X, Delannoy M, Egelman EH, Sohn J - Nat Commun (2015)

Bottom Line: The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers.The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC.Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine Baltimore, Maryland 21205, USA.

ABSTRACT
AIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2(PYD)) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

No MeSH data available.


Related in: MedlinePlus

AIM2FL and isolated AIM2Hin bind dsDNA in a length-dependent manner.(a) Binding of AIM2FL and (b) isolated AIM2Hin to each FAM-labelled dsDNA (1.5 nM) was determined by fluorescence anisotropy. The determined KD values are listed in Supplementary Tables 2–4. (c) Competition binding assays using FAM-dsVACV72 (1.5 nM, 0.06 μg ml−1) and AIM2FL (70 nM) at 400 mM KCl against various dsDNA fragments; the lines are fits to a competition binding equation: 1/(1+((DNAcompetitor)/IC50)Hill constant). The determined IC50 values are listed in Supplementary Tables 5 and 7. (d) Competition binding assays using FAM-dsVACV72 (5 nM, 0.2 μg ml−1) and AIM2Hin (250 nM) at 160 mM KCl against various DNA fragments. The determined values are listed in Supplementary Table 6. The plots of the binding efficiency versus the length of dsDNA for AIM2FL (e) and AIM2Hin (f). The binding efficiency was determined by normalizing the mean IC50 of each fragment to that of dsDNA600, and the data were fit to the Hill equation (the Hill constant for (e) is 4.2±0.2 and (f) is 3.7±0.3; ±indicates s.d., n≥3). The ‘threshold' oligomer is defined as the size of dsDNA (AIM2 cluster) required to exit the apparent lag phase, and the ‘optimal' oligomer is the size of dsDNA (AIM2 cluster) required to reach the inflection point.
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f2: AIM2FL and isolated AIM2Hin bind dsDNA in a length-dependent manner.(a) Binding of AIM2FL and (b) isolated AIM2Hin to each FAM-labelled dsDNA (1.5 nM) was determined by fluorescence anisotropy. The determined KD values are listed in Supplementary Tables 2–4. (c) Competition binding assays using FAM-dsVACV72 (1.5 nM, 0.06 μg ml−1) and AIM2FL (70 nM) at 400 mM KCl against various dsDNA fragments; the lines are fits to a competition binding equation: 1/(1+((DNAcompetitor)/IC50)Hill constant). The determined IC50 values are listed in Supplementary Tables 5 and 7. (d) Competition binding assays using FAM-dsVACV72 (5 nM, 0.2 μg ml−1) and AIM2Hin (250 nM) at 160 mM KCl against various DNA fragments. The determined values are listed in Supplementary Table 6. The plots of the binding efficiency versus the length of dsDNA for AIM2FL (e) and AIM2Hin (f). The binding efficiency was determined by normalizing the mean IC50 of each fragment to that of dsDNA600, and the data were fit to the Hill equation (the Hill constant for (e) is 4.2±0.2 and (f) is 3.7±0.3; ±indicates s.d., n≥3). The ‘threshold' oligomer is defined as the size of dsDNA (AIM2 cluster) required to exit the apparent lag phase, and the ‘optimal' oligomer is the size of dsDNA (AIM2 cluster) required to reach the inflection point.

Mentions: We then tested whether the dsDNA-binding affinity of AIM2 changes with the size of dsDNA, because such a behaviour is expected when ligand binding is dependent on oligomerization1323. For instance, the apparent binding affinity would increase nonlinearly up to the ‘optimal' oligomer dictated by nucleic acid sizes2328. To characterize the DNA-binding property of AIM2FL within the detection limit of our instruments, we used the reported salt concentration-dependent binding of AIM2Hin (ref. 32) and performed binding assays with various dsDNA sizes at 400 mM KCl (Fig. 2). Even in this high salt condition, AIM2FL robustly bound FAM-dsDNA72 (Fig. 2a; the binding constant did not change with FAM-dsDNA72 concentrations, see Supplementary Fig. 1d). Importantly, AIM2FL bound the larger dsDNA more tightly (Fig. 2a), suggesting that oligomerization is integral to dsDNA binding. However, MBP-AIM2FL, MBP-AIM2Hin and AIM2Hin all showed no detectable binding in this high salt condition (Supplementary Fig. 1e), again supporting the positive role of AIM2PYD in dsDNA binding. In 160-mM KCl, isolated AIM2Hin also bound the larger dsDNA more tightly (Fig. 2b). Finally, both AIM2FL and isolated AIM2Hin bound the footprint-size dsDNA (10 bp) with minimal affinity (Supplementary Fig. 1f), suggesting that oligomerization is important for high affinity binding of both AIM2 variants (the dsDNA-binding footprint of AIM2Hin is ∼9 bp32 and that of AIM2FL is ∼12 bp, Supplementary Fig. 1g).


Assembly-driven activation of the AIM2 foreign-dsDNA sensor provides a polymerization template for downstream ASC.

Morrone SR, Matyszewski M, Yu X, Delannoy M, Egelman EH, Sohn J - Nat Commun (2015)

AIM2FL and isolated AIM2Hin bind dsDNA in a length-dependent manner.(a) Binding of AIM2FL and (b) isolated AIM2Hin to each FAM-labelled dsDNA (1.5 nM) was determined by fluorescence anisotropy. The determined KD values are listed in Supplementary Tables 2–4. (c) Competition binding assays using FAM-dsVACV72 (1.5 nM, 0.06 μg ml−1) and AIM2FL (70 nM) at 400 mM KCl against various dsDNA fragments; the lines are fits to a competition binding equation: 1/(1+((DNAcompetitor)/IC50)Hill constant). The determined IC50 values are listed in Supplementary Tables 5 and 7. (d) Competition binding assays using FAM-dsVACV72 (5 nM, 0.2 μg ml−1) and AIM2Hin (250 nM) at 160 mM KCl against various DNA fragments. The determined values are listed in Supplementary Table 6. The plots of the binding efficiency versus the length of dsDNA for AIM2FL (e) and AIM2Hin (f). The binding efficiency was determined by normalizing the mean IC50 of each fragment to that of dsDNA600, and the data were fit to the Hill equation (the Hill constant for (e) is 4.2±0.2 and (f) is 3.7±0.3; ±indicates s.d., n≥3). The ‘threshold' oligomer is defined as the size of dsDNA (AIM2 cluster) required to exit the apparent lag phase, and the ‘optimal' oligomer is the size of dsDNA (AIM2 cluster) required to reach the inflection point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525163&req=5

f2: AIM2FL and isolated AIM2Hin bind dsDNA in a length-dependent manner.(a) Binding of AIM2FL and (b) isolated AIM2Hin to each FAM-labelled dsDNA (1.5 nM) was determined by fluorescence anisotropy. The determined KD values are listed in Supplementary Tables 2–4. (c) Competition binding assays using FAM-dsVACV72 (1.5 nM, 0.06 μg ml−1) and AIM2FL (70 nM) at 400 mM KCl against various dsDNA fragments; the lines are fits to a competition binding equation: 1/(1+((DNAcompetitor)/IC50)Hill constant). The determined IC50 values are listed in Supplementary Tables 5 and 7. (d) Competition binding assays using FAM-dsVACV72 (5 nM, 0.2 μg ml−1) and AIM2Hin (250 nM) at 160 mM KCl against various DNA fragments. The determined values are listed in Supplementary Table 6. The plots of the binding efficiency versus the length of dsDNA for AIM2FL (e) and AIM2Hin (f). The binding efficiency was determined by normalizing the mean IC50 of each fragment to that of dsDNA600, and the data were fit to the Hill equation (the Hill constant for (e) is 4.2±0.2 and (f) is 3.7±0.3; ±indicates s.d., n≥3). The ‘threshold' oligomer is defined as the size of dsDNA (AIM2 cluster) required to exit the apparent lag phase, and the ‘optimal' oligomer is the size of dsDNA (AIM2 cluster) required to reach the inflection point.
Mentions: We then tested whether the dsDNA-binding affinity of AIM2 changes with the size of dsDNA, because such a behaviour is expected when ligand binding is dependent on oligomerization1323. For instance, the apparent binding affinity would increase nonlinearly up to the ‘optimal' oligomer dictated by nucleic acid sizes2328. To characterize the DNA-binding property of AIM2FL within the detection limit of our instruments, we used the reported salt concentration-dependent binding of AIM2Hin (ref. 32) and performed binding assays with various dsDNA sizes at 400 mM KCl (Fig. 2). Even in this high salt condition, AIM2FL robustly bound FAM-dsDNA72 (Fig. 2a; the binding constant did not change with FAM-dsDNA72 concentrations, see Supplementary Fig. 1d). Importantly, AIM2FL bound the larger dsDNA more tightly (Fig. 2a), suggesting that oligomerization is integral to dsDNA binding. However, MBP-AIM2FL, MBP-AIM2Hin and AIM2Hin all showed no detectable binding in this high salt condition (Supplementary Fig. 1e), again supporting the positive role of AIM2PYD in dsDNA binding. In 160-mM KCl, isolated AIM2Hin also bound the larger dsDNA more tightly (Fig. 2b). Finally, both AIM2FL and isolated AIM2Hin bound the footprint-size dsDNA (10 bp) with minimal affinity (Supplementary Fig. 1f), suggesting that oligomerization is important for high affinity binding of both AIM2 variants (the dsDNA-binding footprint of AIM2Hin is ∼9 bp32 and that of AIM2FL is ∼12 bp, Supplementary Fig. 1g).

Bottom Line: The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers.The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC.Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine Baltimore, Maryland 21205, USA.

ABSTRACT
AIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2(PYD)) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

No MeSH data available.


Related in: MedlinePlus