Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3'-phosphoadenosine 5'-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases.

3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5'-phosphosulfate (APS) and PPi.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions.

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca) ion channels represents the activation of the Ca-sensor protein STIM1 upon Ca store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex.

Conserved Dynamic Mechanism of Allosteric Response to L-arg in Divergent Bacterial Arginine Repressors.

Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in and , the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of ArgR.

Sequential activation of STIM1 links Ca with luminal domain unfolding.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca sensing within the endoplasmic reticulum and activation of the store-operated Ca channel Orai1, enabling plasma-membrane Ca influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca ions.

Mechanistic insights into the Orai channel by molecular dynamics simulations.

Highly Ca selective channels trigger a large variety of cellular signaling processes in both excitable and non-excitable cells. Among these channels, the Orai channel is unique in its activation mechanism and its structure. It mediates Ca influx into the cytosol with an extremely small unitary conductance over longer time-scales, ranging from minutes up to several hours.

Crystal structure of a novel domain of the motor subunit of the Type I restriction enzyme EcoR124 involved in complex assembly and DNA binding.

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I.

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation.

Communication between N terminus and loop2 tunes Orai activation.

Ca release-activated Ca (CRAC) channels constitute the major Ca entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca-selective Orai channel and the Ca-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating.

Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription.

The channel Orai1 requires Ca store depletion in the endoplasmic reticulum and an interaction with the Ca sensor STIM1 to mediate Ca signaling. Alterations in Orai1-mediated Ca influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants.

The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation.

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR.

pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I.

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.

Quantum Calculations Indicate Effective Electron Transfer between FMN and Benzoquinone in a New Crystal Structure of Escherichia coli WrbA.

Unlabelled: Quantum mechanical calculations using the Marcus equation are applied to compare the electron-transfer probability for two distinct crystal structures of the Escherichia coli protein WrbA, an FMN-dependent

Nad(p)h: quinone oxidoreductase, with the bound substrate benzoquinone. The calculations indicate that the position of benzoquinone in a new structure reported here and solved at 1.33 Å resolution is more likely to be relevant for the physiological reaction of WrbA than a previously reported crystal structure in which benzoquinone is shifted by ∼5 Å.

Cholesterol modulates Orai1 channel function.

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents.

A calcium-accumulating region, CAR, in the channel Orai1 enhances Ca(2+) permeation and SOCE-induced gene transcription.

The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations.

Influence of ligand binding on structure and thermostability of human α1-acid glycoprotein.

Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β-barrel are described.

Solution NMR and molecular dynamics reveal a persistent alpha helix within the dynamic region of PsbQ from photosystem II of higher plants.

The extrinsic proteins of photosystem II of higher plants and green algae PsbO, PsbP, PsbQ, and PsbR are essential for stable oxygen production in the oxygen evolving center. In the available X-ray crystallographic structure of higher plant PsbQ residues S14-Y33 are missing. Building on the backbone NMR assignment of PsbQ, which includes this "missing link", we report the extended resonance assignment including side chain atoms.

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation.

Resonance assignment of PsbP: an extrinsic protein from photosystem II of Spinacia oleracea.

PsbP (23 kDa) is an extrinsic eukaryotic protein of photosystem II found in the thylakoid membrane of higher plants and green algae. It has been proven to be indispensable for proper functioning of the oxygen evolving complex. By interaction with other extrinsic proteins (PsbQ, PsbO and PsbR), it modulates the concentration of two cofactors of the water splitting reaction, Ca(2+) and Cl(-).

Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.

Background: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.

Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein.

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry.

Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains.

Binding-competent states for L-arginine in E. coli arginine repressor apoprotein.

Arginine repressor of E. coli is a multifunctional hexameric protein that provides feedback regulation of arginine metabolism upon activation by the negatively cooperative binding of L-arginine. Interpretation of this complex system requires an understanding of the protein's conformational landscape.

Inhibition of GlcNAc-processing glycosidases by C-6-azido-NAG-thiazoline and its derivatives.

NAG-thiazoline is a strong competitive inhibitor of GH20 β-N-acetyl- hexosaminidases and GH84 β-N-acetylglucosaminidases. Here, we focused on the design, synthesis and inhibition potency of a series of new derivatives of NAG-thiazoline modified at the C-6 position. Dimerization of NAG-thiazoline via C-6 attached triazole linkers prepared by click chemistry was employed to make use of multivalency in the inhibition.

Expression, characterization and homology modeling of a novel eukaryotic GH84 β-N-acetylglucosaminidase from Penicillium chrysogenum.

β-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal β-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family.