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Journal of molecular biology [journal]
- Chromatin dynamics during differentiation of myeloid cells. [REVIEW]
- J Mol Biol 2014 Aug 26.
Cellular commitment to differentiation requires a tightly synchronized, spatial-temporal interaction of regulatory proteins with the basic DNA and chromatin. A complex network of mechanisms, involving induction of lineage instructive transcription factors, installation or removal of histone modifications and changes in the DNA methylation pattern locally orchestrate the three dimensional chromatin structure and determine cell fate. Maturation of myeloid lineages from haematopoietic stem cells has emerged as a powerful model to study those principles of chromatin mechanisms in cellular differentiation and lineage fate selection. This review summarizes recent knowledge and puts forward novel ideas on how dynamics in the epigenetic landscape of myeloid cells shape the development, immune-activation and leukaemic transformation outcome.
- A Structural Portrait of the PDZ Domain Family. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 22.
PDZ (PSD-95/Discs-large/ZO-1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signalling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side chain and main chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide to understanding the structural basis for the diverse specificities across the family.
- DNA-recognition by a σ(54) transcriptional activator from Aquifex aeolicus. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 22.
Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsible for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.
- The Enigmatic Cytoplasmic Regions of KCNH Channels. [REVIEW]
- J Mol Biol 2014 Aug 22.
KCNH channels are expressed across a vast phylogenetic and evolutionary spectrum. In humans they function in a wide range of tissues and serve as biomarkers and targets for diseases such as cancer and cardiac arrhythmias. These channels share a general architecture with other voltage-gated ion channels but are distinguished by the presence of an N-terminal Per-Arnt-Sim (PAS) domain and a C-terminal domain with homology to cyclic nucleotide binding domains (referred to as the CNBh domain). Cytosolic regions outside these domains show little conservation between KCNH families but within a family are strongly conserved across species, likely reflecting variability that confers specificity to individual channel types. PAS and CNBh domains participate in channel gating, but at least twice in evolutionary history the PAS domain has been lost, and in one family it is omitted by alternate transcription to create a distinct channel subunit. In this focused review we present current knowledge of the structure and function of these cytosolic regions, discuss their evolution as modular domains, and provide our perspective on the important questions moving forward.
- The PHD finger of p300 influences its ability to acetylate histone and non-histone targets. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 22.
In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, while it enhanced p300 self-acetylation. A Rubinstein-Taybi syndrom-related point mutation in the p300 PHD finger resulted in increased self-acetylation, but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin.
- Bacterial voltage-gated sodium channels (BacNaVs) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart. [REVIEW]
- J Mol Biol 2014 Aug 22.
Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts.
- Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 19.
Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from the high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for a differential binding of the γ-subunit to the GAFa domains of the α- and β-subunit are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.
- Coevolution of specificity determinants in eukaryotic glutamyl- and glutaminyl-tRNA synthetases. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 18.
The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA(Gln) for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA(Gln) and tRNA(Glu) with glutamate. This ancient GluRS also separately differentiated to exclude tRNA(Gln) as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA(Gln) and tRNA(Glu) recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model, and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p-GluRS complex upon the diauxic shift to respiratory conditions.
- Ryanodine Receptors: Allosteric Ion Channel Giants. [REVIEW]
- J Mol Biol 2014 Aug 15.
The endoplasmic (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca(2+) stores. Ryanodine Receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release the Ca(2+) upon triggering. With molecular weights exceeding 2.2 MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with the tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy. Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscle. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca(2+) in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolution, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.
- DNA Looping Provides for "Intersegmental Hopping" by Proteins: A Mechanism for Long-Range Site Localization. [JOURNAL ARTICLE]
- J Mol Biol 2014 Aug 15.
Studies of how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~100 Dam copies per cell) and the regulated methylation of closely spaced sites which controls the expression of virulence genes in several human pathogens. Provocatively, we find Dam travels between its recognition sites most efficiently when those sites are ~500 base pairs apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), invoking recent reports that demonstrate DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.