-defensins are cyclic octadecapeptides encoded by the modified ␣-defensin genes of certain nonhuman primates. The recent demonstration that human ␣-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of -defensins on Bacillus anthracis (Sterne). We tested rhesus -defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of -defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of -defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that -defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.Under normal circumstances Bacillus anthracis causes human infections only in individuals exposed to infected farm animals or their spore-contaminated products. The virulence of B. anthracis primarily derives from the hardiness of its spores, an anti-phagocytic capsule that surrounds its vegetative cells (1), and two secreted binary toxins: lethal toxin (LeTx) 3 and edema toxin (EdTx). Both toxins contain protective antigen (PA, 83 kDa). LeTx also contains lethal factor (LF, 90 kDa), and EdTx contains edema factor (EF, 89 kDa). The genes for all three toxin components, PA, LF, and EF, reside on the pXO1 plasmid (2), and those responsible for capsule synthesis exist on the pXO2 plasmid (3). Both of these plasmids are required for in vivo virulence (3).EF is an adenylate cyclase (4) and LF is a zinc-dependent metalloprotease that selectively attacks certain MAPK kinases (5, 6). PA is required to allow both of the other toxin components to enter host cells (7). When PA binds a cellular receptor (8), it is cleaved into PA63 (63 kDa) and PA20 (20 kDa). The PA20 diffuses away, and the residual receptor-bound PA63 molecules self-associate into ring-shaped heptamers (9) that bind EF or LF with high affinity (10 -12). Oligomerization of PA63 leads to endocytosis, which transports the complexes to an acidic compartment (13-15). Here, the heptameric pre-pore changes into an integral-membrane pore (16, 17) that translocates EF or LF into the cytosol (18). Immu...
Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.
Alpha-(1-->2)-mannosidase I from the endoplasmic reticulum (ERManI), a Family 47 glycoside hydrolase, is a key enzyme in the N-glycan synthesis pathway. Catalytic-domain crystal structures of yeast and human ERMan1s have been determined, the former with a hydrolytic product and the latter without ligands, with the inhibitors 1-deoxymannojirimycin and kifunensine, and with a thiodisaccharide substrate analog. Both inhibitors were bound at the base of the funnel-shaped active site as the unusual 1C4 conformer, while the substrate analog glycon is a 3S1 conformer. In the current study, AutoDock was used to dock alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranose with its glycon in chair (1C4,4C1), half-chair (3H2,3H4,4H3), skew-boat (OS2,3S1,5S1), boat (2,5B,3,OB,B1,4,B2,5), and envelope (3E,4E,E3,E4) conformations into the yeast ERManI active site. Both docked energies and forces on docked ligand atoms were calculated to determine how the ligand distorts to the transition state. From these, we can conclude that (1) both 1C4 and OS2 can be the starting conformers; (2) the most likely binding pathway is 1C4-->3H2-->OS2-->3,OB-->3S1-->3E; (3) the transition state is likely to be close to a 3E conformation.
Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.
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