Structural Insights into the Assembly of CARMA1 and BCL10

<div><p>The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 Å. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.</p> </div

Mutational analysis of the Mal-TIR domain.

<p>(<b>A</b>) Selective mutants of solvent-exposed residues that are highly conserved across different species and used in the NF-κB reporter assay. (<b>B</b>) Alanine scanning of each residue of the AB loop (except alanine and glycine) for the NF-κB reporter assay. Black bars indicate decreased (less than 50%) NF-κB activity. RLU: relative luciferase unit; Luc: firefly luciferase activity; Ren: Renilla luciferase activity. GST pull-down assay for the MyD88-TIR and Mal-TIR variants (<b>C</b>) and for the TLR4-TIR and Mal-TIR variants (<b>D</b>). MyD88-TIR and TLR4-TIR were purified as GST fusion proteins. Mal-TIR was double-tagged with His₆ and Myc and purified with Ni-NTA. The resulting complexes were analyzed by SDS-PAGE and Western blotting. (<b>E</b>) Bar graph of Mal-TIR wild type and mutants (E108A and F117A) binding to MyD88-TIR (left) or TLR4-TIR (right). The error bars indicate the standard error of the mean (n = 3 separate experiments). * indicates a P value<0.05, ** indicates a P value<0.001.</p

Data collection and refinement statistics.

a<p>the highest resolution shell.</p>b<p>.</p>c<p>.</p>d<p><b><i>R</i></b>_free, calculated the same as <b><i>R</i></b>_crystal, but from a test set containing 5% of data excluded from the refinement calculation.</p

The binding surface of CARMA1-CARD.

<p>(<b>A</b>) The representative basic residues (R35, K41, K69 and R72) on the positive surface of the CARMA1-CARD are colored blue. (<b>B</b>) Interactions between the basic residues and surrounding sulfate ions. The side chains of basic residues R35, K41, K69 and R72 and sulfate ions are shown as sticks. The oxygen atoms and sulfur atoms are colored red and yellow, respectively. Hydrogen bonds are shown as red dashed lines. (<b>C</b>) Sequence alignment of CARMA1-CARD proteins from different species. The conserved amino acids are highlighted in red. Conserved residues in the basic patch are denoted with asterisks.</p

Crystal structure of Mal-TIR.

<p>(<b>A</b>) Schematic illustration of five TIR-domain-containing adaptors involved in TLR signaling. Mal: MyD88 adaptor-like protein; MyD88: myeloid differentiation factor 88; TRIF: TIR-domain-containing adaptor-inducing interferon-β; TRAM: TRIF-related adaptor molecule; SARM: sterile and HEAT/armadillo (ARM) motif protein; DD: death domain; ID: intermediate domain; SAM: sterile α-motif. (<b>B</b>) Cartoon representation of the structure of Mal-TIR. The β-strands are shown in pink, the α-helices in cyan and the connecting loops in salmon. The AB loop is colored red. The red dotted line represents the region that is not resolved in this structure.</p

Biochemical characteristics of wild-type and mutant Mal-TIR domains.

<p>(<b>A</b>) Structural comparison between wild-type and mutant Mal-TIR domains. Crystal structures of the wild-type (cyan), D96N (magenta) and S180L (yellow) mutants of the Mal-TIR domain are shown. The residues Asp96 and Ser180 in the wild type as well as Asn96 in D96N and Leu180 in S180L are labeled. Nitrogen and oxygen atoms in the side chains are colored blue and red, respectively. (<b>B,D</b>) Electrostatic surfaces of wild type and the D96N mutant or the S180L mutant. Surfaces are colored by electrostatic potential ranging from red (−10 k_bT/e_c) to blue (+10 k_bT/e_c), where k_b is the Boltzmann constant, T is temperature and e_c is the electron charge. The electrostatic potentials were calculated by solving the Poisson-Boltzmann equation with APBS plugin of PyMol program (DeLano Scientific LLC). (<b>C,E</b>) The molecular surfaces of wild-type (left) and the D96N mutant (right) or the S180L mutant of the Mal-TIR domains. Carbon, nitrogen, oxygen and sulfur atoms are colored yellow, blue, red and orange, respectively. (<b>F</b>) GST pull-down assay for the interaction of wild-type Mal-TIR and mutants with TLR4-TIR and MyD88-TIR.</p

Data collection and refinement statistics.

a<p>the highest resolution shell.</p>b<p>.</p>c<p><b><i>R</i></b>_crystal = .</p>d<p><b><i>R</i></b>_free, calculated the same as <b><i>R</i></b>_crystal, but from a test set containing 5% of data excluded from the refinement calculation.</p

The binding surface of BCL10-CARD.

<p>(<b>A</b>) Superposition of homology models of BCL10-CARD. The computational results from the programs MODELLER and SWISS-MODEL are represented with green and blue cartoon models, respectively. (<b>B</b>) The electrostatic surface of BCL10-CARD. Red: negative; blue: positive; and white: neutral. Residues E50, E53 and E54 are labeled. (<b>C</b>) Sequence alignment of BCL10-CARD proteins from different species. The conserved amino acids are highlighted with red, and the conserved acidic residues that make up the acidic patch are denoted with asterisks. (<b>D</b>) Protein docking models of the CARMA1-CARD and BCL10-CARD calculated using the ZDOCK server. CARMA1-CARD and BCL10-CARD are colored magenta and gray, respectively. Three out of ten best scoring complexes place the BCL10-CARD domain approaching the interface containing residues R35, K41, K69 and R72 of CARMA1-CARD. The side chains of residues R35, K41, K69 and R72 are shown as sticks. (<b>E</b>) The interactions between CARD domains in the best docking complex model. CARMA1-CARD and BCL10-CARD are colored magenta and gray, respectively. Side chains of E50, E53 and E54 of BCL10 as well as R53, K41, K69 and R72 of CARMA1 are shown as stick.</p

Association of CARMA1 and BCL10.

<p>(<b>A</b>) Co-IP analysis of interactions between BCL10-CARD and variants of CARMA1-CARD. HEK293T cells were transiently co-transfected with GFP-tagged BCL10-CARD and wild type or mutants of Myc-tagged CARMA1-CARD constructs. Cell extracts were immunoprecipitated using an anti-Myc antibody and blotted using anti-GFP. (<b>B</b>) Co-IP analysis of interactions between Myc-tagged CARMA1-CARD and wild type or mutants of GFP-tagged BCL10-CARD constructs. (<b>C,D</b>) Bar graph displaying interactions between CARMA1-CARD and BCL10-CARD. (<b>E</b>) The effect of wild type and mutants of CARMA1 on the NF-κB reporter assay. (<b>F</b>) The effect of wild type and mutants of BCL10 on NF-κB activity. RLU: relative luciferase unit; Luc: firefly luciferase activity; and Ren: Renilla luciferase activity. The error bars indicate the standard error of the mean (n = 3 separate experiments). * indicates a P value<0.05, ** indicates a P value<0.001. (<b>G, H</b>) The expression levels of CARMA1 and BCL10 in NF-κB assays were checked by immunoblotting with anti-GFP, anti-MYC and anti-GAPDH antibodys, respectively.</p

Data collection and refinement statistics.

a<p>the highest resolution shell.</p>b<p> </p>c<p><b><i>R</i></b>_crystal = </p>d<p><b><i>R</i></b>_free, calculated the same as <b><i>R</i></b>_crystal, but from a test set containing 5% of data excluded from the refinement calculation.</p