The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition.

Phospholipase C gamma 2 (PLCγ2) plays important roles in cell signaling downstream of various membrane receptors. PLCγ2 contains a multidomain inhibitory region critical for its regulation, while it has remained unclear how these domains contribute to PLCγ2 activity modulation. Here we determined three structures of human PLCγ2 in autoinhibited states, which reveal dynamic interactions at the autoinhibition interface, involving the conformational flexibility of the Src homology 3 (SH3) domain in the inhibitory region, and its previously unknown interaction with a carboxyl-terminal helical domain in the core region.

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor.

Crystal structures and biochemical analyses of the bacterial arginine dihydrolase ArgZ suggests a "bond rotation" catalytic mechanism.

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.

Structural basis for transcription initiation by bacterial ECF σ factors.

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σ (σ-RNAP) at 2.

Structural insights into the unique mechanism of transcription activation by Caulobacter crescentus GcrA.

Canonical bacterial transcription activators bind to non-transcribed promoter elements to increase transcription of their target genes. Here we report crystal structures of binary complexes comprising domains of Caulobacter crescentus GcrA, a noncanonical bacterial transcription factor, that support a novel mechanism for transcription activation through the preferential binding of methylated cis-regulatory elements and the promotion of open complex formation through an interaction with region 2 of the principal σ factor, σ70. We present crystal structures of the C-terminal, σ factor-interacting domain (GcrA-SID) in complex with domain 2 of σ70 (σ702), and the N-terminal, DNA-binding domain (GcrA-DBD) in complex with methylated double-stranded DNA (dsDNA).

A Quantitative Analysis of Brain Soluble Tau and the Tau Secretion Factor.

Neurofibrillary tangles (NFTs) represent products of insoluble tau protein in the brains of patients with Alzheimer disease (AD). The cerebrospinal fluid (CSF) tau level is a biomarker in AD diagnosis. The soluble portion of tau protein in brain parenchyma is presumably the source for CSF tau but this has not previously been quantified.

Papain-like protease (PLpro) inhibitory effects of cinnamic amides from Tribulus terrestris fruits.

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria.

Structural basis of a novel activity of bacterial 6-pyruvoyltetrahydropterin synthase homologues distinct from mammalian 6-pyruvoyltetrahydropterin synthase activity.

Escherichia coli 6-carboxytetrahydropterin synthase (eCTPS), a homologue of 6-pyruvoyltetrahydropterin synthase (PTPS), possesses a much stronger catalytic activity to cleave the side chain of sepiapterin in vitro compared with genuine PTPS activity and catalyzes the conversion of dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin in vivo. Crystal structures of wild-type apo eCTPS and of a Cys27Ala mutant eCTPS complexed with sepiapterin have been determined to 2.3 and 2.

Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) from Synechococcus sp. PCC 7942.

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacterium Synechococcus sp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method.

Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis.

Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei.

Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility.

Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis.

Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.

Crystallization and preliminary X-ray data analysis of a DJ-1 homologue from Arabidopsis thaliana (AtDJ-1D).

A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.

Structural insights into the dual substrate specificities of mammalian and Dictyostelium dihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin.

Up to now, d-threo-tetrahydrobiopterin (DH(4), dictyopterin) was detected only in Dictyostelium discoideum, while the isomer L-erythro-tetrahydrobioterin (BH(4)) is common in mammals. To elucidate the mechanism of DH(4) regeneration by D. discoideum dihydropteridine reductase (DicDHPR), we have determined the crystal structure of DicDHPR complexed with NAD(+) at 2.

Purification, crystallization and crystallographic analysis of Dictyostelium discoideum phenylalanine hydroxylase in complex with dihydrobiopterin and FeIII.

Dictyostelium discoideum phenylalanine hydroxylase (DicPAH; residues 1-415) was expressed in Escherichia coli and purified for structural analysis. Apo DicPAH and DicPAH complexed with dihydrobiopterin (BH(2)) and Fe(III) were crystallized using 0.06 M PIPES pH 7.

Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum.

Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources.