Qingqiu Huang

Envelope stress is a trigger of CRISPR RNA‐mediated DNA silencing in Escherichia coli

A widespread feature in the genomes of most bacteria and archaea is an array of clustered, regularly interspaced short palindromic repeats (CRISPRs) that, together with a group of CRISPR‐associated (Cas) proteins, mediate immunity against invasive nucleic acids such as plasmids and viruses. Here, the CRISPR‐Cas system was activated in cells expressing a plasmid‐encoded protein that was targeted to the twin‐arginine translocation (Tat) pathway. Expression of this Tat substrate resulted in upregulation of the Cas enzymes and subsequent silencing of the encoding plasmid in a manner that required the BaeSR two‐component regulatory system, which is known to respond to extracytoplasmic stress. Furthermore, we confirm that the CasCDE enzymes form a stable ternary complex and appear to function as the catalytic core of the Cas system to process CRISPR RNA into its mature form. Taken together, our results indicate that the CRISPR‐Cas system targets DNA directly as part of a defence mechanism in bacteria that is overlapping with but not limited to phage infection.

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

Background: Cas2 is universally conserved and essential for new CRISPR spacer acquisition. Results: Bha_Cas2 uses a single metal ion to cleave dsDNA and is likely activated by a pH-dependent conformational change. A method to classify Cas2 into ssRNase and dsDNase is proposed. Conclusion: B. halodurans and T. thermophilus Cas2 are metal-dependent endonucleases. Significance: dsDNase activity is consistent with the direct involvement of Cas2 in new spacer acquisition. The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg2+ or Mn2+), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop.

Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom

A snake-venom protein named AHP-LAAO has been purified from Agkistrodon halys pallas venom using four-stage chromatography. AHP-LAAO is a novel member of the snake-venom L-amino-acid oxidase family. Its amino-acid sequence shows high homology to other members of this family. For L-leucine, the values of k(cat) and K(M) are 31.1 s(-1) and 0.25 mM, respectively. The molecular weight of AHP-LAAO is about 60.7 kDa as determined by MALDI-TOF mass spectrometry. AHP-LAAO can also induce apoptosis of cultured Hela cells. Two sets of diffraction data with similar resolution limits (about 2.5 A) were collected independently at MacCHESS (Cornell High Energy Synchrotron Source, USA) and IHEP (Institute of High Energy Physics, Beijing, China). The crystals belong to space group I2(1)3, with unit-cell parameter a = 169.31 A, corresponding to one molecule in the asymmetric unit and a volume-to-weight ratio of 3.33 A(3) Da(-1). The final structural model is similar to that of L-amino-acid oxidase from Calloselasma rhodostoma venom.

The crystal structure of a novel, inactive, lysine 49 PLA2 from Agkistrodon acutus venom: an ultrahigh resolution, AB initio structure determination

The crystal structure of acutohaemolysin, a lysine 49 phospholipase A2 protein with 1010 non-hydrogen protein atoms and 232 water molecules, has been determined ab initio using the program SnB at an ultrahigh resolution of 0.8 Å. The lack of catalytic activity appears to be related to the presence of Phe102, which prevents the access of substrate to the active site. The substitution of tryptophan for leucine at residue 10 interferes with dimer formation and may be responsible for the additional loss of hemolytic activity. The ultrahigh resolution of the experimental diffraction data permits alternative conformations to be modeled for disordered residues, many hydrogen atoms to be located, the protonation of the Nϵ2 atom in the catalytic residue His48 to be observed experimentally, and the density of the bonding electrons to be analyzed in detail.

Structural Basis for the Interaction between the Growth Factor-binding Protein GRB10 and the E3 Ubiquitin Ligase NEDD4.

In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel β-sheet composed of the F β-strand of GRB10 SH2 and the C β-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.

Nucleic acid binding surface and dimer interface revealed by CRISPR-associated CasB protein structures

TfuCasB1 and TfuCasB1 bind by molecular sieving (View Interaction: 1, 2)

Purification, partial characterization and crystallization of acucetin, a protein containing both disintegrin-like and cysteine-rich domains released by auto-proteolysis of a P-III-type metalloproteinase AaH-IV from Agkistrodon acutus venom.

AaH-IV, a P-III-type metalloproteinase found in Agkistrodon acutus venom, readily cleaves itself to release a stable protein named acucetin at 310 K under neutral and weakly alkaline conditions. A partial amino-acid residue sequence of acucetin indicates that the protein has a high homology to snake-venom proteins containing both disintegrin-like and cysteine-rich domains. Acucetin has been crystallized in space group R32, with hexagonal unit-cell parameters a = b = 155.98, c = 76.07 A. The V(M) value of about 2.97 A(3) Da(-1) suggests the presence of only one molecule in the asymmetric unit.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of two cysteine-rich secretory proteins from Naja atra and Trimeresurus stejnegeri venoms

Cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals and snake venoms. They possess apparent homology but varying functions. The structure of CRISPs has remained elusive. Two novel members of the family, natrin and stecrisp, have been purified from Naja atra and Trimeresurus stejnegeri venoms, respectively. Their crystals diffract X-rays to resolution limits of 2.1 and 1.6 angstroms, respectively, and belong to the orthorhombic system with different space groups, unit-cell parameters and numbers of molecules per asymmetric unit. Their structures will contribute a structural basis for further functional studies of this family.

Design and evaluation of novel glutaminase inhibitors.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

SNAP‐25 is also an iron–sulfur protein

SNAP‐25 has a cysteine cluster located at its linker domain. In vivo, the cysteine residues in this cluster can be palmitoylated, and the hydrophobic palmitate molecules can target SNAP‐25 to the presynaptic membrane. Here, we report that the SNAP‐25a expressed in Escherichia coli is also an iron–sulfur protein binding an iron–sulfur cluster using the cysteine residues in its cysteine cluster. Therefore, SNAP‐25a uses the same cysteine residues to bind two different prosthetic groups (iron–sulfur cluster and palmitate). Because the binding sites of these two prosthetic groups overlap, we suggest that these two modifications occur at different times, and probably at different places in the cell.