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

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

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

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

MOESM1 of Engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate

Additional file 1: Table S1. Primers used in this study. Table S2. Introduction of alcohol acyltransferases from plants and the titer of geranyl acetate. Fig. S1. Plasmid maps and DNA sequences

MOESM1 of Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae

Additional file 1: Table S1. The sequence of various promoter. Table S2. The sequence of various terminators. Table S3. L sequence. Table S4. The integration locus of chromosome. Table S5. PCR primers used in this work. Table S6. The sequence of integration locus (site2). Figure S1. The diagrams of promoter plasmids (Circular Display). Figure S2. The diagrams of terminator plasmids (linear display). Figure S3. The diagrams of integration locus (site1) plasmids. Figure S4. The transformants of β-carotene on SC-Ura solid medium

Additional file 2 of Chromosome constitution and genetic relationships of Morus spp. revealed by genomic in situ hybridization

Additional file 2: Supplementary Table 1. List of the mulberry accessions used in this study

The Inhibitory Helix Controls the Intramolecular Conformational Switching of the C-Terminus of STIM1

<div><p>Store-operated Ca²⁺ entry (SOCE) is a critical Ca²⁺ signaling pathway in many cell types. After sensing Ca²⁺ store depletion in the endoplasmic reticulum (ER) lumen, STIM1 (STromal Interaction Molecule 1) oligomerizes and then interacts with and activates the Orai1 calcium channel. Our previous research has demonstrated that the inhibitory helix (IH) adjacent to the first coiled-coil region (CC1) of STIM1 may keep the whole C-terminus of STIM1 in an inactive state. However, the specific conformational change of CC1-IH that drives the transition of STIM1 from the resting state to the active state remains elusive. Herein, we report the structural analysis of CC1-IH, which revealed that the entire CC1-IH molecule forms a very long helix. Structural and biochemical analyses indicated that IH, and not the CC1 region, contributes to the oligomerization of STIM1. Small-angle X-ray scattering (SAXS) analysis suggested that the C-terminus of STIM1 including the IH region displays a collapsed conformation, whereas the construct without the IH region has an extended conformation. These two conformations may correspond to the conformational states of the C-terminus of STIM1 before and after activation. Taken together, our results provide direct biochemical evidence that the IH region controls the conformational switching of the C-terminus of STIM1.</p> </div

Different conformational states of STIM1-Ccyto in solution as revealed by SAXS.

<p>(<b>A</b>, <b>B</b>) Oligomeric states of STIM1-CIS (<b>A</b>) and STIM1-CIS-DelIH (<b>B</b>) in solution determined using multi-angle laser scattering. (<b>C</b>, <b>D</b>) Experimental scattering curves for STIM1-CIS (<b>C</b>) and STIM1-CIS-DelIH (<b>D</b>) in solution: 1, experimental SAXS curve; 2, scattering patterns calculated from the DAMMIN model; 3, smooth curve back transformed from p(r) and extrapolated to a zero scattering angle for STIM1-CIS. Upper-right, the distance distribution function p(r) computed using the program GNOM. (<b>E</b>) Hypothetical molecular shape of STIM1-CIS in solution. STIM1-CIS may adopt a collapsed conformation, which represents the resting state of STIM1-Ccyto. The SOAR and CC1-IH dimers are shown in light pink and cyan, respectively. (<b>F</b>) Superimposition of an STIM1-CIS bead model (shown as white dots) developed based on the SAXS data and the known SOAR structure (α-helices shown in light pink). (<b>G</b>) Hypothetical molecular shape of STIM1-CIS-DelIH in solution. STIM1-CIS-DelIH may adopt a stretched and elongated conformation when IH is removed (or when IH releases the SOAR dimer), which represents the activated state of STIM1-Ccyto. The SOAR and CC1-IH dimers are shown in light pink and cyan, respectively. (<b>H</b>) Superimposition of the STIM1-CIS-DelIH bead model (shown as white dots) developed based on the SAXS data and the known SOAR and CC1 structures (α-helices shown in light pink and cyan, respectively).</p