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The 8 most recent public experiments

Defining the interaction of perforin with calcium and the phospholipid membrane Daouda A.K. Traore, James C Whisstock Download data as .tar

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca2 + -dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studiesrevealthatCa2+ bindingtriggersaconformationalchange in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforin- like) domain, indicating that subsequent monomer–monomer interactions at the membrane surface are required for perforin pore formation. Publication: Biochem J.

This is a test experiment.

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. These datasets are derivatives of the peripheral stalk from T.thermophilus A-ATPase. Native crystals were soaked in Lutetium(III) acetate (2K7c_3_###.img) and Dysprosium(III) chloride (2K3#######.img). Resulting maps were used to create the pdb model 3V6I. The model was used to identify bending and twisting motions inherent within the structure that accommodate movements within the ATPase.

Structure-Informed Design of an Enzymatically Inactive Vaccine Component for Group A Streptococcus Anna Henningham, Daniel J. Ericsson, Karla Langer, Lachlan Casey, Blagojce Jovcevski, G. Singh Chhatwal, J. Andrew Aquilina, Michael R. Batzloff, Bostjan Kobe, Mark Walker Download data as .tar

Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4.

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts. This work was funded by Lottery Health Research (CJS; grant number 265027), the Auckland Medical Research Foundation (JUF and CJS; grant number 1110004. JUF; grant number 1109008), the National eScience Infrastructure (JUF), and the Maurice Wilkins Centre for Molecular Biodiscovery Flexible Research Seeding Programme (JUF and CJS). We further acknowledge salary support from the Maurice Wilkins Centre for Molecular Biodiscovery (CJS, JUF) and Summer Studentship funding from the Faculty of Science, University of Auckland (CJS, RMT).

Structure of the heterodimer of human NONO and paraspeckle protein component 1 Charlie Bond , Daniel Passon , Mihwa Lee Download data as .tar

Proteins of the Drosophila behavior/human splicing (DBHS) family include mammalian SFPQ (PSF), NONO (p54nrb), PSPC1, and invertebrate NONA and Hrp65. DBHS proteins are predominately nuclear, and are involved in transcriptional and posttranscriptional gene regulatory functions as well as DNA repair. DBHS proteins influence a wide gamut of biological processes, including the regulation of circadian rhythm, carcinogenesis, and progression of cancer. Additionally, mammalian DBHS proteins associate with the architectural long noncoding RNA NEAT1 (Menε/β) to form paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNA. Here we describe the crystal structure of the heterodimer of the multidomain conserved region of the DBHS proteins, PSPC1 and NONO. These proteins form an extensively intertwined dimer, consistent with the observation that the different DBHS proteins are typically copurified from mammalian cells, and suggesting that they act as obligate heterodimers. The PSPC1/NONO heterodimer has a right-handed antiparallel coiled-coil that positions two of four RNA recognition motif domains in an unprecedented arrangement on either side of a 20-Å channel. This configuration is supported by a protein:protein interaction involving the NONA/paraspeckle domain, which is characteristic of the DBHS family. By examining various mutants and truncations in cell culture, we find that DBHS proteins require an additional antiparallel coiled-coil emanating from either end of the dimer for paraspeckle subnuclear body formation. These results suggest that paraspeckles may potentially form through self-association of DBHS dimers into higher-order structures.

Synthesis of new (-)-Bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase. Geetha Velmourougane, Michael B. Harbut, Seema Dalal, Sheena McGowan, Christine A. Oellig, James C. Whisstock, Michael Klemba, Doron C. Greenbaum Download data as .tar

The essential malarial PfA-M1 metalloaminopeptidase is a validated drug target that functions in the terminal stages of hemoglobin digestion. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1 and provides an excellent scaffold for the development of novel research tools as well as more effective PfA-M1 inhibitors. Here we present a new, efficient and high yielding protocol for the synthesis of bestatin-derivatives from commercially available natural and unnatural N-Boc-D-amino acids. We developed a diverse library of bestatin derivatives with variants at the sidechain of either the α-hydroxy-β-amino acid or the adjacent natural α-amino acid. Surprisingly we found that large aromatic rings at the P1 position resulted in potent inhibition against PfA-M1, while small hydrophobic sidechains were favored at the P1’ position. These data contrast previous studies that suggested the primary substrate specificity (S1) pocket of the PfA-M1 enzyme is unable to accommodate side-chains much larger than a P1 phenylalanine. To understand these apparently contradictory data, we determined the X-ray crystal structure of the PfA-M1 / bestatin-Tyr(OBzl) complex. The structure revealed a substantial inhibitor-induced rearrangement of the primary loop that forms the S1 pocket that permits accommodation of the bestatin-Tyr(OBzl) inhibitor. These findings are in contrast to most proteases where the S1 pocket is considered to define primary enzyme specificity through substantial rigidity. Taken together, our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme, which may eventually be of therapeutic value for the treatment of malaria.</abstract> To cite this data use the following DOI: 10.4225/52/557FAD81B7777

X-ray crystal structure of the streptococcal specific phage lysin PlyC Sheena McGowan, Ashley Buckle, James Whisstock Download data as .tar

Publication (PNAS)

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

This entry contains two diffraction datasets:

Automatically generated on 2015-06-02 06:39:07 by To cite this data use the following DOI: 10.4225/52/557FAA5E63100