Defining the interaction of perforin with calcium and the phospholipid membrane

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

X-ray crystal structure of the fibrinolysis inhibitor {alpha}2-antiplasmin (2R9Y)

<div>The serpin alpha(2)-antiplasmin (SERPINF2) is the principal inhibitor of plasmin and inhibits fibrinolysis. Accordingly, alpha(2)-antiplasmin deficiency in humans results in uncontrolled fibrinolysis and a bleeding disorder. alpha(2)-antiplasmin is an unusual serpin, in that it contains extensive N- and C-terminal sequences flanking the serpin domain. The N-terminal sequence is crosslinked to fibrin by factor XIIIa, whereas the C-terminal region mediates the initial interaction with plasmin. To understand how this may happen, we have determined the 2.65A X-ray crystal structure of an N-terminal truncated murine alpha(2)-antiplasmin. The structure reveals that part of the C-terminal sequence is tightly associated with the body of the serpin. This would be anticipated to position the flexible plasmin-binding portion of the C-terminus in close proximity to the serpin Reactive Center Loop where it may act as a template to accelerate serpin/protease interactions.</div><div><br></div><p></p

X-ray crystal structure of the streptococcal specific phage lysin PlyC

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

The 1.6 A Crystal Structure of the Catalytic Domain of PlyB, a Bacteriophage Lysin Active Against Bacillus anthracis. (2NW0)

<div>Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.</div><div><br></div><p></p

The Crystal Structure of the Transthyretin-like Protein from Salmonella dublin, a Prokaryote 5-Hydroxyisourate Hydrolase (2GPZ)

<div>The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.</div><div><br></div><p></p

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation. (2H4R)

<div>Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.</div><div><br></div><p></p

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase (3EBG, 3EBH, 3EBI)

This experiment holds 3EBG, 3EBH and 3EBI

The structure of granzyme C reveals an unusual mechanism of protease auto-inhibition (3FZZ)

<div>Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-A X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the active-site serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu-Glu motif at positions 192-193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-A structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone.</div><div><br></div><p></p

P. Baehni, professeur en médecine dentaire

P. Baehni, professeur en médecine dentair

Additional file 3: Figure S3. of Brinp1 −/− mice exhibit autism-like behaviour, altered memory, hyperactivity and increased parvalbumin-positive cortical interneuron density

Pyramidal neuron distribution in the adult Brinp1 −/− somatosensory neocortex. a Normal density of NeuN+ cells in the Brinp1 −/− somatosensory neocortex. b No significant change in distribution of NeuN+ cells through somatosensory cortical layers, F(1,6) = 1.423, p = 0.278, repeat measures two-way ANOVA. Sections counterstained with DAPI. c No significant changes in Cux1 cell number or distribution were detected in the Brinp1 −/− somatosensory neocortex, F(1,6) = 2.027, p = 0.204, repeat measures two-way ANOVA. N = 4 WT, 4 Brinp1 −/− mice. *p < 0.05, **p < 0.01. All data represented as the mean ± SD. (PNG 2569 kb