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Background: Phosphatidylethanolamine-binding protein (PEBP) is a basic protein found in numerous tissues from a wide range of species. The screening of gene and protein data banks defines a family of PEBP-related proteins that are present in a variety of organisms, including Drosophila and inferior eukaryotes. PEBP binds to phosphatidylethanolamine and nucleotides in vitro, but its biological function...
Background: 1 H and 15 N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both 1 H and 15 N holds great promise for the elucidation of functionally...
In their natural environment, three-dimensional structures of proteins undergo significant fluctuations and are often partially or completely disordered. This phenomenon recently became the focus of much attention, as many proteins, especially from higher organisms, were shown to contain large intrinsically disordered regions. Such disordered regions may become ordered only under very specific circumstances,...
The human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp)...
Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed...
Cryo-electron microscopy (cryo-EM) studies of the bacteriophage ϕ29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base,...
The constant attack on DNA by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. Visualizing the effects of these lesions on the structure of duplex DNA is key to understanding their biologic consequences. The most definitive method of obtaining such structures, X-ray crystallography, is troublesome to employ owing to the difficulty...
We present a method for the computer-based iterative assembly of native-like tertiary structures of helical proteins from α-helical fragments. For any pair of helices, our method, called MATCHSTIX, first generates an ensemble of possible relative orientations of the helices with various ways to form hydrophobic contacts between them. Those conformations having steric clashes, or a large radius of...
Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the...
Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure...
The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites...
RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 Å. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled...
We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-Å-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of...
Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between...
The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with...
IL-22 is an IL-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. IL-22 biological activity is initiated by binding to a cell-surface complex composed of IL-22R1 and IL-10R2 receptor chains and further regulated by interactions with a soluble binding protein, IL-22BP, which shares sequence similarity with an extracellular region...
Sirtuin enzymes comprise a unique class of NAD + -dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally...
Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here...
Peptides comprising residues 106–126 of the human prion protein (PrP) exhibit many features of the full-length protein. PrP(106–126) induces apoptosis in neurons, forms fibrillar aggregates, and can mediate the conversion of native cellular PrP (PrP C ) to the scrapie form (PrP Sc ). Despite a wide range of biochemical and biophysical studies on this peptide, including investigation...
Human autoimmune regulator (AIRE) functions to control thymic expression of tissue-specific antigens via sequence-specific histone H3 recognition by its plant homeodomain (PHD) finger. Mutations in the AIRE PHD finger have been linked to autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report the three-dimensional solution structure of the first PHD finger of human...
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