Chromodomains read the arginine code of post-translational targeting.

Chromodomains typically recruit protein complexes to chromatin and read the epigenetic histone code by recognizing lysine methylation in histone tails. We report the crystal structure of the chloroplast signal recognition particle (cpSRP) core from Arabidopsis thaliana, with the cpSRP54 tail comprising an arginine-rich motif bound to the second chromodomain of cpSRP43. A twinned aromatic cage reads out two neighboring nonmethylated arginines and adapts chromodomains to a non-nuclear function in post-translational targeting

Conformational landscape and active conformation of the Dcp1:Dcp2 mRNA decapping complex

Enzymes are dynamic molecular machines. Many insights into the molecular details of their function have been gained from crystal structures. But in the case of highly dynamic enzymes crystal structures are prone to packing artifacts. They also hide dynamic information that is often crucial for the understanding of the enzymatic function. A striking example is the bilobed decapping enzyme Dcp2. It catalyzes the removal of the protecting 5’ cap from eukaryotic mRNAs and thereby regulates gene expression. The activity of the C-terminal catalytic domain (CD) of Dcp2 is increased in a stepwise manner by the N-terminal regulatory domain (RD) and the activator proteins Dcp1 and Edc1. The two domains of Dcp2 are connected by a flexible linker and Dcp2 has been shown to be highly dynamic. Several crystal structures of Dcp2 in the free state and in complex with these activator proteins have been solved. These structures can be divided into six groups with vastly different domain orientations. Due to the dynamic nature of Dcp2 it is challenging to determine whether the obtained crystal structures are adopted in solution. This explains why the mechanisms of activation and the structure of the catalytically active state of the enzyme still remain controversial despite this wealth of structural information. To address these questions we explore the conformations that Dcp2 samples in solution using a suite of methyl TROSY based NMR experiments. By combining CSP, 13 C-CPMG relaxation dispersion, NOESY and PRE experiments we show that Dcp2 samples three different structural states in solution: an open and a closed conformation and a catalytically active form. The apo and the activator bound enzyme complexes exchanges between catalytically impaired open and closed conformations. Substrate binding to the Dcp1:Dcp2 complex competes with the closed conformation and results in a highly dynamic assembly. The stable catalytically active state of the decapping complex is only formed in the presence of substrate and both activators, which is explained by a novel crystal structure of the quaternary complex. In summary, we provide a detailed model of how the conformational landscape of Dcp2 is modulated by decapping activators and how this increases the catalytic activity

Simulation of PRE data for flexible ATCUN and TEMPO spin labels in decapping complexes

Proteins are inherently dynamic entities. While many functional aspects can be derived from their static or time- averaged structures, a comprehensive mechanistic understanding often requires knowledge about the specific dynamics underlying a biological process. NMR is a powerful technique to study these dynamics on different timescales ranging from picoseconds up to seconds. Different NMR methods can be exploited to investigate structural changes such as side chain rotations, molecular tumbling, folding and domain motions, which accompany binding events, allostery or catalysis. Dcp2 is a Nudix enzyme which catalyzes the removal of the 5' protecting cap structure from mRNA, resulting in an efficient termination of gene expression. The catalytic activity is already present in the isolated catalytic domain (CD), but it is incrementally increased by the N-terminal regulatory domain (NRD) and the activator proteins Dcp1 and Edc1. Although a number of crystal structures of Dcp2 decapping complexes has been solved, their biological relevance and the mode of activation remain controversial. We used TEMPO and ATCUN spin labels for paramagnetic relaxation enhancement (PRE) measurements on reporter methyl groups to elucidate domain orientations in the absence and presence of activators and substrate. The experimental data could not be fitted by a single spin label conformation, indicating that the spin label is flexible. To account for this, we generated an ensemble of 500 spin label conformations using a simulated annealing workflow in XPLOR-NIH. A combination of three spin label conformations was selected from this ensemble, based on a Monte Carlo approach implemented in Matlab, that minimized the difference between experimental and calculated PRE values. In agreement with published results 1 we find that the correlation between experimental and calculated PRE values does not increase with the use of more than three conformations. The procedure improves the identification of the actually sampled domain orientation in decapping complexes and thereby provides an accurate picture of the structure of the decapping complex in solution

Cell-binding IgM in CSF is distinctive of multiple sclerosis and targets the iron transporter SCARA5.

Intrathecal IgM production in multiple sclerosis (MS) is associated with a worse disease course. To investigate pathogenic relevance of autoreactive IgM in MS, CSF from two independent cohorts, including MS patients and controls, were screened for antibody binding to induced pluripotent stem cell-derived neurons and astrocytes, and a panel of CNS- related cell lines. IgM binding to a primitive neuro-ectodermal tumour cell line discriminated 10% of MS donors from controls. Transcriptomes of single IgM producing CSF B cells from patients with cell-binding IgM were sequenced and used to produce recombinant monoclonal antibodies for characterisation and antigen identification. We produced 5 cell-binding recombinant IgM antibodies, of which one, cloned from an HLA-DR + plasma-like B cell, mediated antigen-dependent complement activation. Immunoprecipitation and mass spectrometry, and biochemical and transcriptome analysis of the target cells identified the iron transport scavenger protein SCARA5 as the antigen target of this antibody. Intrathecal injection of a SCARA5 antibody led to an increased T cell infiltration in an EAE model. CSF IgM might contribute to CNS inflammation in MS by binding to cell surface antigens like SCARA5 and activating complement, or by facilitating immune cell migration into the brain