Structural Model of the mIgM B-Cell Receptor Transmembrane Domain From Self-Association Molecular Dynamics Simulations

Antigen binding to B-cell antigen receptors (BCRs) followed by signaling initiates the humoral immune response. The signaling is intimately coupled to nanoclustering of BCRs and their sorting to specific membrane domains, a process that is ruled by interactions between the BCR transmembrane domain and lipids. While the structure of the extracellular domains of BCRs has been resolved, little is known about the configuration of the constituting four immunoglobulin domains spanning the membrane. Here, we modeled the structure of the transmembrane (TM) domain of the IgM B-cell receptor using self-assembly coarse-grained molecular dynamics simulations. The obtained quaternary structure was validated against available experimental data and atomistic simulations. The IgM-BCR-TM domain configuration shows a 1:1 stoichiometry between the homodimeric membrane-bound domain of IgM (mIgM) and a Ig-α/Ig-β heterodimer. The mIgM homodimer is based on an asymmetric association of two mIgM domains. We show that a specific site of the Ig-α/Ig-β heterodimer is responsible for the association of IgM-BCRs with lipid rafts. Our results further suggest that this site is blocked in small-sized IgM-BCR clusters. The BCR TM structure provides a molecular basis for the previously suggested dissociation activation model of B-cell receptors. Self-assembly molecular dynamics simulations at the coarse-grained scale here proved as a versatile tool in the study of receptor complexes

Closeup view of the effector-binding site of DasR-EBD.

<p>The stereo views show the interaction of DasR-EBD with the α-anomeric configuration of (a) GlcN-6-P and (b) GlcNAc-6-P. GlcN-6-P, GlcNAc-6-P and the interacting protein residues are presented as stick models and water molecules are depicted as red spheres. In the sugar molecules, the phosphor, oxygen, nitrogen and carbon atoms are coloured in yellow, red, dark blue and grey, respectively.</p

High diversity in the positioning of the DBDs observed among the GntR/HutC transcription factors.

<p>Superposition of the crystal structures of ligand-free SauR from <i>S</i>. <i>avermitilis</i> (DBD in grey, PDB-ID 3EET), ligand-free DasR from <i>S</i>. <i>coelicolor</i> (blue, PDB-ID 4ZS8), GlcNAc-6-P-bound NagR from <i>B</i>. <i>subtilis</i> (light green, PDB-ID 4U0W), ligand-free PhnF from <i>M</i>. <i>smegmatis</i> (light yellow, PDB-ID 3F8M), sulphate-bound NagR (dark green, PDB-ID 2WV0), ligand-free YydK from <i>B</i>. <i>subtilis</i> (light brown, PDB-ID 3BWG) and NagR in complex with palindromic dsDNA (olive, PDB-ID 4WWC). The prior listing correlates with the clockwise display of the DBDs of the various proteins starting with the DBD of SauR (in grey) at the top of the figure. The superimposed EBDs of the dimeric repressors are located in the centre and rendered transparent. The superimposed EBDs show the following r.m.s. deviations between Cα-positions when compared to the EBD (residues 87–252, chain A) of ligand-free DasR: 1.76 Å (ligand-free SauR), 2.08 Å (GlcNAc-6-P-bound NagR), 1.47 Å (ligand-free PhnF), 2.13 Å (sulphate-bound NagR), 1.94 Å (ligand-free YydK) and 1.65 Å (DNA-bound NagR).</p

A conformational selection model best describes the allosteric regulation of GntR/HutC transcription factors.

<p>The population density of distinct functional states is schematically compared to the conformational diversity observed in GntR/HutC crystal structures. The different functional states are (I) effector- and DNA-free repressor, (II) DNA-bound repressor and (III) effector-bound repressor. Effector-bound repressors cannot bind DNA in the conformations depicted in (IV) and (V).</p

A comparison of ligand-free DasR with the GlcNAc-6-P- and DNA-bound structures of NagR.

<p>(a) and (b) Superposition of dimers of ligand-free DasR (blue) and GlcNAc-6-P-bound NagR (green) displayed in a side view (a) and a top view (b). The centres of mass of the DBDs in each dimer were calculated with CHIMERA and are presented as red spheres. The DBDs of DasR and NagR can be oriented identically after applying a rotation of about 140° to either one of the DBDs around the axes indicated in grey (grey rods) and as calculated with program CHIMERA (138°, left DBDs and 142°, right DBDS). (c) Superposition of a ligand-free DasR dimer (blue) with the crystal structure of NagR-DBDs (green) in complex with DNA (orange). Helix α_E6 of chain A of DasR is coloured in orange red. The helix axes (red rods) of the DNA-recognition helices α_D3 were calculated with CHIMERA.</p

Structure of ligand-free DasR-EBD.

<p>(a) Crystal structure of a ligand-free DasR-EBD dimer. Only one monomer is contained in the asymmetric unit (shown in blue) and a second, symmetry-related monomer is shown in transparent white. (b) Closeup view of the effector-binding site and the N-terminal interdomain linker in ligand-free DasR-EBD. Residues 91–97 of the linker segment are shown as stick model with the corresponding <i>2F</i>_<i>o</i><i>-F</i>_<i>c</i> map (grey mesh) contoured at 1 σ to highlight the well-defined conformation of the main chain of this segment.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Structure of DasR-EBD in complex with GlcN-6-P.

<p>(a) Crystal structure of dimeric DasR-EBD bound to GlcN-6-P in a cartoon representation. The monomers are coloured in blue and light grey, while GlcN-6-P is shown as a stick model. (b) Topology plot of monomeric DasR-EBD in complex with GlcN-6-P. Secondary structure elements are displayed as blue cylinders (α-helices) and arrows (β-strands). The linker segment between the DBD and EBD is highlighted in bold. Due to the highly similar overall conformation, the GlcNAc-6-P-bound DasR-EBD structure is not shown.</p

Structure and topology plot of ligand-free full-length DasR.

<p>(a) DasR dimer without DNA or a potential effector molecule with the monomers coloured in blue and light grey. (b) Topology plot of full-length DasR in the ligand-free state. Secondary structure elements are displayed as blue cylinders (α-helices) and arrows (β-strands). The linker segment between the DBD and EBD is highlighted in bold. (c) Closeup view of the linker segment between the DBD and EBD in ligand-free DasR. Residues 86–96 of the linker segment are shown as stick model with the corresponding <i>2F</i>_<i>o</i><i>-F</i>_<i>c</i> electron density (grey mesh) contoured at 1 σ to emphasize the well-ordered secondary structure in this region.</p