Crystal structure of the GDP-bound human M-RAS protein in two crystal forms.

M-RAS plays a crucial role in the RAF-MEK signaling pathway. When activated by GTP, M-RAS forms a complex with SHOC2 and PP1C, initiating downstream RAF-MEK signal transduction. In this study, the crystal structure of the GDP-bound human M-RAS protein is presented with two forms of crystal packing.

Enhancing the apo protein tyrosine phosphatase non-receptor type 2 crystal soaking strategy through inhibitor-accessible binding sites.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) has recently been recognized as a promising target for cancer immunotherapy. Despite extensive structural and functional studies of other protein tyrosine phosphatases, there is limited structural understanding of PTPN2. Currently, there are only five published PTPN2 structures and none are truly unbound due to the presence of a mutation, an inhibitor or a loop (related to crystal packing) in the active site.

Application of sulfur SAD to small crystals with a large asymmetric unit and anomalous substructure.

The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al.

The structure of the cysteine-rich region from human histone-lysine N-methyltransferase EHMT2 (G9a).

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein-protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms.

Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy.

Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.

Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA.

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography.

Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution.

Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics.

Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A.

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440.

Cryo-EM structures of vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.

Human gastric pathogen () is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown.

Crystal structure of the major quadruplex formed in the promoter region of the human c-MYC oncogene.

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds.

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits.

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A-D). We describe highly potent ()-5-[()-2-amino-2-carboxyethyl]-4,5-dihydro-1-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors.

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c.

We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland, L. J., et al.

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate.

Structural Basis for Negative Allosteric Modulation of GluN2A-Containing NMDA Receptors.

NMDA receptors mediate excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. Here, we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo state of the GluN1 ligand-binding domain (LBD), which is incapable of triggering channel gating. We describe structural determinants of NAM binding in crystal structures of the GluN1/2A LBD heterodimer, and analyses of NAM-bound LBD structures corresponding to active and inhibited receptor states reveal a molecular switch in the modulatory binding site that mediate the allosteric inhibition.

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability.

The hypothesis that the recent rapid evolution of primate cytochromes c, which primarily involves residues in the least stable Ω-loop (Ω-loop C, residues 40-57), stabilizes the heme crevice of cytochrome c relative to other mammals, is tested. To accomplish this goal, we have compared the properties of human and spider monkey cytochrome c and a set of four variants produced in the process of converting human cytochrome c into spider monkey cytochrome c. The global stability of all variants has been measured by guanidine hydrochloride denaturation.

Structure of a mitochondrial cytochrome c conformer competent for peroxidase activity.

At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule.

Inhibitors of membranous adenylyl cyclases.

Membranous adenylyl cyclases (mACs) constitute a family of nine isoforms with different expression patterns. Studies with mAC gene knockout mice provide evidence for the notion that AC isoforms play distinct (patho)physiological roles. Consequently, there is substantial interest in the development of isoform-selective mAC inhibitors.

Impact of divalent metal ions on regulation of adenylyl cyclase isoforms by forskolin analogs.

Mammalian membranous adenylyl cyclases (mACs) play an important role in transmembrane signalling events in almost every cell and represent an interesting drug target. Forskolin (FS) is an invaluable research tool, activating AC isoforms 1-8. However, there is a paucity of AC isoform-selective FS analogs.

Structure-activity relationships for the interactions of 2'- and 3'-(O)-(N-methyl)anthraniloyl-substituted purine and pyrimidine nucleotides with mammalian adenylyl cyclases.

Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2',3'-(O)-(N-methylanthraniloyl) (MANT)-substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5.

Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2',3'-o-(N-methylanthraniloyl)-inosine 5'-triphosphate.

2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP.

The C1 homodimer of adenylyl cyclase binds nucleotides with high affinity but possesses exceedingly low catalytic activity.

Membranous adenylyl cyclase (AC) subtypes play differential roles in the regulation of cell functions. The C1- and C2-subunits of AC form a heterodimer that efficiently catalyzes cAMP formation and constitutes a very useful model system for AC analysis at a molecular level. Intriguingly, C1 and C2 homodimers exist, too.