Expression, purification and preliminary crystallographic analysis of recombinant human small glutamine-rich tetratricopeptide-repeat protein

The production and crystallization of the tetratricopeptide-repeat domain of human small glutamine-rich tetratricopeptide-repeat protein are reported. A 2.4 Å native diffraction data set has been obtained

Expression, purification, crystallization and preliminary X-ray analysis of full-length human RIG-I

The human innate immune system can detect invasion by microbial pathogens through pattern-recognition receptors that recognize structurally conserved pathogen-associated molecular patterns. Retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) are one of the two major families of pattern-recognition receptors that can detect viral RNA. RIG-I, belonging to the RLH family, is capable of recognizing intracellular viral RNA from RNA viruses, including influenza virus and Ebola virus. Here, full-length human RIG-I (hRIG-I) was cloned in Escherichia coli and expressed in a recombinant form with a His-SUMO tag. The protein was purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.85 Å resolution; the crystal belonged to space group F23, with unit-cell parameters a = b = c = 216.43 Å, [alpha] = [beta] = [gamma] = 90°.Published versio

Brigadoon - 21

Scene from the Wright State University Theatre Department\u27s production of Brigadoon by Alan Jay Lerner and Frederick Loewe. This production was performed during the 1978-1979 school year.https://corescholar.libraries.wright.edu/theatre_brigadoon/1004/thumbnail.jp

Insights from the Structure of an Active Form of <i>Bacillus thuringiensis</i> Cry5B

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112–698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27–698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112–698) structure, and also exhibits a change in the conformation of residues 112–140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112–140 in its alternate α-helix conformation. Finally, based on the Cry5B(27–698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity

The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus

RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5Bbinding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79–303) of DDX5 contains the typical features found in the structures of other DEADbox helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52–78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1–51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.Web of Scienc

The C-Terminal Segment of the Cysteine-Rich Interdomain of Plasmodium falciparum Erythrocyte Membrane Protein 1 Determines CD36 Binding and Elicits Antibodies That Inhibit Adhesion of Parasite-Infected Erythrocytes▿

Attachment of erythrocytes infected by Plasmodium falciparum to receptors of the microvasculature is a major contributor to the pathology and morbidity associated with malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is expressed at the surface of infected erythrocytes and is linked to both antigenic variation and cytoadherence. PfEMP-1 contains multiple adhesive modules, including the Duffy binding-like domain and the cysteine-rich interdomain region (CIDR). The interaction between CIDRα and CD36 promotes stable adherence of parasitized erythrocytes to endothelial cells. Here we show that a segment within the C-terminal region of CIDRα determines CD36 binding specificity. Antibodies raised against this segment can specifically block the adhesion to CD36 of erythrocytes infected with various parasite strains. Thus, small regions of PfEMP-1 that determine binding specificity could form suitable components of an antisequestration malaria vaccine effective against different parasite strains

Songs

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix α3 shifting position initially, followed by movement of α2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species