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Novel disulfide bond-mediated dimerization of the CARD domain was revealed by the crystal structure of CARMA1 CARD.

Jang TH, Park JH, Park HH - PLoS ONE (2013)

Bottom Line: Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available.Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å.Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan, South Korea.

ABSTRACT
CARMA1, BCL10 and MALT1 form a large molecular complex known as the CARMA1 signalosome during lymphocyte activation. Lymphocyte activation via the CARMA1 signalosome is critical to immune response and linked to many immune diseases. Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available. Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å. Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.

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Related in: MedlinePlus

Formation of dimeric CARMA1 CARD in vitro.A. Gel-filtration chromatography showed that CARMA1 CARD forms a dimeric and further oligoemric states without reducing agent (black line). The dimeric form of CARMA1 CARD became monometic in response to the addition of reducing agent (red line). C28A mutant also became monomeric in solution. B. Native-PAGE confirmed that CARMA1 CARD exists as dimer in solution and became a monomer by adding DTT. The position of the dimer form and monomeric form on native-PAGE are indicated. The amounts of DTT added are shown above the picture. w/o: without DTT. +: with DTT. C. Determination of the molecular mass of the CARMA1 CARD in the absence of reducing agent by multi-angle light scattering. D. Determination of the molecular mass of the CARMA1 CARD in the presence of reducing agent (5 mM DTT) by multi-angle light scattering. E. Determination of the molecular mass of C28A mutant by multi-angle light scattering.
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pone-0079778-g003: Formation of dimeric CARMA1 CARD in vitro.A. Gel-filtration chromatography showed that CARMA1 CARD forms a dimeric and further oligoemric states without reducing agent (black line). The dimeric form of CARMA1 CARD became monometic in response to the addition of reducing agent (red line). C28A mutant also became monomeric in solution. B. Native-PAGE confirmed that CARMA1 CARD exists as dimer in solution and became a monomer by adding DTT. The position of the dimer form and monomeric form on native-PAGE are indicated. The amounts of DTT added are shown above the picture. w/o: without DTT. +: with DTT. C. Determination of the molecular mass of the CARMA1 CARD in the absence of reducing agent by multi-angle light scattering. D. Determination of the molecular mass of the CARMA1 CARD in the presence of reducing agent (5 mM DTT) by multi-angle light scattering. E. Determination of the molecular mass of C28A mutant by multi-angle light scattering.

Mentions: Disulfide bond-mediated oligomerization was also detected in solution. Despite its complexity, dimeric (even highly oligomeric) and monomeric CARMA1 CARD co-exists in solution without reducing agent dithiothreitol (DTT), which breaks the disulfide bond. However, gel-filtration chromatography revealed that the addition of 5 mM DTT resulted in CARMA1 CARD becoming monomeric (Figure 3A). C28A mutant also was not able to produce oligomeric peaks on the profile of gel-filtration chromatography. The reducing agent mediated monomeric change of CARMA1 CARD was also detected upon native-PAGE analysis. Specifically, dimeric CARMA1 CARD became the monomeric form in a DTT concentration dependent manner (Figure 3B). To confirm the previous results, we calculated the absolute molecular weight of CARMA1 CARD with or without reducing agent using multi-angle light scattering (MALS). C28A mutant was also measured with MALS. The calculated monomeric molecular weight of CARMA1 CARD including the C-terminal His-tag was 12.35 kDa, while that of MALS was 22.18 kDa (0.7% fitting error) without DTT and 17.12 kDa with 5 mM DTT (Figure 3C and 3D). C28A was 14.21 kDa (Figure 3E). As shown in Figure 3C, CARMA1 CARD exists as dimerand further oligomer mixture in solution and was all changed to the monomeric form by the addition of DTT and by mutating C28 to Alanine, indicating that CARMA1 CARD forms a stable dimer in solution and that the dimerization is mediated by a disulfide bond.


Novel disulfide bond-mediated dimerization of the CARD domain was revealed by the crystal structure of CARMA1 CARD.

Jang TH, Park JH, Park HH - PLoS ONE (2013)

Formation of dimeric CARMA1 CARD in vitro.A. Gel-filtration chromatography showed that CARMA1 CARD forms a dimeric and further oligoemric states without reducing agent (black line). The dimeric form of CARMA1 CARD became monometic in response to the addition of reducing agent (red line). C28A mutant also became monomeric in solution. B. Native-PAGE confirmed that CARMA1 CARD exists as dimer in solution and became a monomer by adding DTT. The position of the dimer form and monomeric form on native-PAGE are indicated. The amounts of DTT added are shown above the picture. w/o: without DTT. +: with DTT. C. Determination of the molecular mass of the CARMA1 CARD in the absence of reducing agent by multi-angle light scattering. D. Determination of the molecular mass of the CARMA1 CARD in the presence of reducing agent (5 mM DTT) by multi-angle light scattering. E. Determination of the molecular mass of C28A mutant by multi-angle light scattering.
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Related In: Results  -  Collection

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pone-0079778-g003: Formation of dimeric CARMA1 CARD in vitro.A. Gel-filtration chromatography showed that CARMA1 CARD forms a dimeric and further oligoemric states without reducing agent (black line). The dimeric form of CARMA1 CARD became monometic in response to the addition of reducing agent (red line). C28A mutant also became monomeric in solution. B. Native-PAGE confirmed that CARMA1 CARD exists as dimer in solution and became a monomer by adding DTT. The position of the dimer form and monomeric form on native-PAGE are indicated. The amounts of DTT added are shown above the picture. w/o: without DTT. +: with DTT. C. Determination of the molecular mass of the CARMA1 CARD in the absence of reducing agent by multi-angle light scattering. D. Determination of the molecular mass of the CARMA1 CARD in the presence of reducing agent (5 mM DTT) by multi-angle light scattering. E. Determination of the molecular mass of C28A mutant by multi-angle light scattering.
Mentions: Disulfide bond-mediated oligomerization was also detected in solution. Despite its complexity, dimeric (even highly oligomeric) and monomeric CARMA1 CARD co-exists in solution without reducing agent dithiothreitol (DTT), which breaks the disulfide bond. However, gel-filtration chromatography revealed that the addition of 5 mM DTT resulted in CARMA1 CARD becoming monomeric (Figure 3A). C28A mutant also was not able to produce oligomeric peaks on the profile of gel-filtration chromatography. The reducing agent mediated monomeric change of CARMA1 CARD was also detected upon native-PAGE analysis. Specifically, dimeric CARMA1 CARD became the monomeric form in a DTT concentration dependent manner (Figure 3B). To confirm the previous results, we calculated the absolute molecular weight of CARMA1 CARD with or without reducing agent using multi-angle light scattering (MALS). C28A mutant was also measured with MALS. The calculated monomeric molecular weight of CARMA1 CARD including the C-terminal His-tag was 12.35 kDa, while that of MALS was 22.18 kDa (0.7% fitting error) without DTT and 17.12 kDa with 5 mM DTT (Figure 3C and 3D). C28A was 14.21 kDa (Figure 3E). As shown in Figure 3C, CARMA1 CARD exists as dimerand further oligomer mixture in solution and was all changed to the monomeric form by the addition of DTT and by mutating C28 to Alanine, indicating that CARMA1 CARD forms a stable dimer in solution and that the dimerization is mediated by a disulfide bond.

Bottom Line: Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available.Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å.Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Graduate School of Biochemistry at Yeungnam University, Gyeongsan, South Korea.

ABSTRACT
CARMA1, BCL10 and MALT1 form a large molecular complex known as the CARMA1 signalosome during lymphocyte activation. Lymphocyte activation via the CARMA1 signalosome is critical to immune response and linked to many immune diseases. Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available. Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å. Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.

Show MeSH
Related in: MedlinePlus