Equilibrium Binding Model for CpG DNA-Dependent Dimerization of Toll-like Receptor 9 Ectodomain.

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.This work was supported by: -US NIH grant R01-GM102869 -Wellcome Trust Senior Research Fellowships 101908/Z/13/Z and 217191/Z/19/Z to Y.M

Crystal structure of the Z-ring associated cell division protein ZapC from Escherichia coli

AbstractBacterial cell division involves a contractile ring that organises downstream proteins at the division site and which contains the tubulin homologue FtsZ. ZapC has been discovered as a non-essential regulator of FtsZ. It localises to the septal ring and deletion of zapC leads to a mild phenotype, while overexpression inhibits cell division. Interference with cell division is facilitated by an interaction with FtsZ. Here, we present the 2.9Å crystal structure of ZapC from Escherichia coli. ZapC forms a dimer and comprises two domains that belong to the Royal superfamily of which many members bind methylated arginines or lysines. ZapC contains an N-terminal chromo-like domain and a Tudor-like C-terminal domain. We show by ITC that ZapC binds the C-terminal tail of FtsZ

Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing.

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.This work was supported by Wellcome Trust through Senior Research Fellowship 101908/Z/13/Z and PhD Studentship 205833/Z/16/Z

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding

Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1-RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding

CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions.

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1.

Funder: Gates Cambridge Trust; Id: http://dx.doi.org/10.13039/501100005370During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly

Multi-Messenger Gravitational Wave Searches with Pulsar Timing Arrays: Application to 3C66B Using the NANOGrav 11-year Data Set

When galaxies merge, the supermassive black holes in their centers may form binaries and, during the process of merger, emit low-frequency gravitational radiation in the process. In this paper we consider the galaxy 3C66B, which was used as the target of the first multi-messenger search for gravitational waves. Due to the observed periodicities present in the photometric and astrometric data of the source of the source, it has been theorized to contain a supermassive black hole binary. Its apparent 1.05-year orbital period would place the gravitational wave emission directly in the pulsar timing band. Since the first pulsar timing array study of 3C66B, revised models of the source have been published, and timing array sensitivities and techniques have improved dramatically. With these advances, we further constrain the chirp mass of the potential supermassive black hole binary in 3C66B to less than $(1.65\pm0.02) \times 10^9~{M_\odot}$ using data from the NANOGrav 11-year data set. This upper limit provides a factor of 1.6 improvement over previous limits, and a factor of 4.3 over the first search done. Nevertheless, the most recent orbital model for the source is still consistent with our limit from pulsar timing array data. In addition, we are able to quantify the improvement made by the inclusion of source properties gleaned from electromagnetic data to `blind' pulsar timing array searches. With these methods, it is apparent that it is not necessary to obtain exact a priori knowledge of the period of a binary to gain meaningful astrophysical inferences.Comment: 14 pages, 6 figures. Accepted by Ap