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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

AIM2PYD is necessary for oligomerization and dsDNA binding in the presence of excess dsDNA.(a) Top: a cartoon demonstrating the rationale of the described FRET experiments. The two differentially coloured ovals represent fluorophore (Dylight-550 and Dylight-650)-labelled AIM2. Bottom: a sample fluorescence emission spectra of an equimolar mixture of FRET donor and acceptor labelled AIM2FL. (b) Changes in the ratio between the FRET donor emission (λmax: 578 nm) and the acceptor emission (λmax: 678 nm) at each indicated dsDNA concentration. The apparent oligomerization constants (KDF) were obtained by fitting the data to a Hill equation and are listed in Supplementary Table 8. (c) A plot of binding efficiency versus the length of dsDNA for AIM2FL. The data were fit to the Hill equation (the Hill constant is 3.2±0.3; ±indicates s.d., n≥3). The efficiency was determined by normalizing the mean KDF of each fragment to that of dsDNA600. (d) The FRET ratio of AIM2Hin with increasing amounts of various dsDNA. The red arrow indicates the concentration of AIM2Hin present in the assay.
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f5: AIM2PYD is necessary for oligomerization and dsDNA binding in the presence of excess dsDNA.(a) Top: a cartoon demonstrating the rationale of the described FRET experiments. The two differentially coloured ovals represent fluorophore (Dylight-550 and Dylight-650)-labelled AIM2. Bottom: a sample fluorescence emission spectra of an equimolar mixture of FRET donor and acceptor labelled AIM2FL. (b) Changes in the ratio between the FRET donor emission (λmax: 578 nm) and the acceptor emission (λmax: 678 nm) at each indicated dsDNA concentration. The apparent oligomerization constants (KDF) were obtained by fitting the data to a Hill equation and are listed in Supplementary Table 8. (c) A plot of binding efficiency versus the length of dsDNA for AIM2FL. The data were fit to the Hill equation (the Hill constant is 3.2±0.3; ±indicates s.d., n≥3). The efficiency was determined by normalizing the mean KDF of each fragment to that of dsDNA600. (d) The FRET ratio of AIM2Hin with increasing amounts of various dsDNA. The red arrow indicates the concentration of AIM2Hin present in the assay.

Mentions: When basal AIM2 encounters foreign dsDNA in the cytoplasm, the individual molecules must assemble into the inflammasome even in the presence of excess binding sites (for example, nearly 400,000 binding sites are in the genome of one F. tularensis). To test this idea, we labelled two separate AIM2FL populations with a Förster resonance energy transfer (FRET) donor and acceptor (Fig. 5a, top). As previously observed for full-length IFI16 (ref. 23), saturating FRET signals were indeed detected from AIM2FL in a dsDNA size-dependent manner even when the substrate is present in excess (Fig. 5a,b; Supplementary Table 8). However, unlike IFI16 that required at least 60-bp dsDNA23, FRET signals were detected from dsDNA fragments as short as 24 bp, suggesting that the minimal binding unit for AIM2 oligomerization is a dimer compared with a tetramer for IFI16. Consistent with our competition assays (Fig. 2), plotting the normalized binding efficiency versus dsDNA length from the FRET assay data also suggests a cooperative relationship in which the binding affinity of AIM2FL can increase as much as 1,000-fold when the size of available dsDNA is 10 times longer (the Hill constant is ∼3; Fig. 5c). Labelled AIM2Hin populations failed to generate FRET signals at 160 mM KCl (Supplementary Fig. 1j,k); however, we detected FRET signals from labelled AIM2Hin if the salt concentration of the reaction buffer was lowered to 60 mM KCl (Fig. 5d). Nevertheless, unlike AIM2FL, FRET signals from labelled AIM2Hin also peaked at the dsDNA concentration equivalent to the amount of AIM2Hin present in these assays, but decreased with excess dsDNA (Fig. 5d). Also, unlike AIM2FL, the peak amplitude was correlated with the size of each dsDNA (Fig. 5d). These observations suggest that the AIM2Hin oligomers are likely different from those assembled AIM2FL, and that AIM2PYD is required for robust dsDNA binding and polymerization in the presence of excess dsDNA. In addition, the observed cooperative relationship between dsDNA size and oligomerization activity (Fig. 5c) is consistent not only with our competition experiments (Fig. 2e) but also with the previous in vivo observation32, thus further supporting our ‘digital ruler' concept in the regulation of the AIM2 inflammasome.


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)

AIM2PYD is necessary for oligomerization and dsDNA binding in the presence of excess dsDNA.(a) Top: a cartoon demonstrating the rationale of the described FRET experiments. The two differentially coloured ovals represent fluorophore (Dylight-550 and Dylight-650)-labelled AIM2. Bottom: a sample fluorescence emission spectra of an equimolar mixture of FRET donor and acceptor labelled AIM2FL. (b) Changes in the ratio between the FRET donor emission (λmax: 578 nm) and the acceptor emission (λmax: 678 nm) at each indicated dsDNA concentration. The apparent oligomerization constants (KDF) were obtained by fitting the data to a Hill equation and are listed in Supplementary Table 8. (c) A plot of binding efficiency versus the length of dsDNA for AIM2FL. The data were fit to the Hill equation (the Hill constant is 3.2±0.3; ±indicates s.d., n≥3). The efficiency was determined by normalizing the mean KDF of each fragment to that of dsDNA600. (d) The FRET ratio of AIM2Hin with increasing amounts of various dsDNA. The red arrow indicates the concentration of AIM2Hin present in the assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525163&req=5

f5: AIM2PYD is necessary for oligomerization and dsDNA binding in the presence of excess dsDNA.(a) Top: a cartoon demonstrating the rationale of the described FRET experiments. The two differentially coloured ovals represent fluorophore (Dylight-550 and Dylight-650)-labelled AIM2. Bottom: a sample fluorescence emission spectra of an equimolar mixture of FRET donor and acceptor labelled AIM2FL. (b) Changes in the ratio between the FRET donor emission (λmax: 578 nm) and the acceptor emission (λmax: 678 nm) at each indicated dsDNA concentration. The apparent oligomerization constants (KDF) were obtained by fitting the data to a Hill equation and are listed in Supplementary Table 8. (c) A plot of binding efficiency versus the length of dsDNA for AIM2FL. The data were fit to the Hill equation (the Hill constant is 3.2±0.3; ±indicates s.d., n≥3). The efficiency was determined by normalizing the mean KDF of each fragment to that of dsDNA600. (d) The FRET ratio of AIM2Hin with increasing amounts of various dsDNA. The red arrow indicates the concentration of AIM2Hin present in the assay.
Mentions: When basal AIM2 encounters foreign dsDNA in the cytoplasm, the individual molecules must assemble into the inflammasome even in the presence of excess binding sites (for example, nearly 400,000 binding sites are in the genome of one F. tularensis). To test this idea, we labelled two separate AIM2FL populations with a Förster resonance energy transfer (FRET) donor and acceptor (Fig. 5a, top). As previously observed for full-length IFI16 (ref. 23), saturating FRET signals were indeed detected from AIM2FL in a dsDNA size-dependent manner even when the substrate is present in excess (Fig. 5a,b; Supplementary Table 8). However, unlike IFI16 that required at least 60-bp dsDNA23, FRET signals were detected from dsDNA fragments as short as 24 bp, suggesting that the minimal binding unit for AIM2 oligomerization is a dimer compared with a tetramer for IFI16. Consistent with our competition assays (Fig. 2), plotting the normalized binding efficiency versus dsDNA length from the FRET assay data also suggests a cooperative relationship in which the binding affinity of AIM2FL can increase as much as 1,000-fold when the size of available dsDNA is 10 times longer (the Hill constant is ∼3; Fig. 5c). Labelled AIM2Hin populations failed to generate FRET signals at 160 mM KCl (Supplementary Fig. 1j,k); however, we detected FRET signals from labelled AIM2Hin if the salt concentration of the reaction buffer was lowered to 60 mM KCl (Fig. 5d). Nevertheless, unlike AIM2FL, FRET signals from labelled AIM2Hin also peaked at the dsDNA concentration equivalent to the amount of AIM2Hin present in these assays, but decreased with excess dsDNA (Fig. 5d). Also, unlike AIM2FL, the peak amplitude was correlated with the size of each dsDNA (Fig. 5d). These observations suggest that the AIM2Hin oligomers are likely different from those assembled AIM2FL, and that AIM2PYD is required for robust dsDNA binding and polymerization in the presence of excess dsDNA. In addition, the observed cooperative relationship between dsDNA size and oligomerization activity (Fig. 5c) is consistent not only with our competition experiments (Fig. 2e) but also with the previous in vivo observation32, thus further supporting our ‘digital ruler' concept in the regulation of the AIM2 inflammasome.

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