Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Changes in the fatty-acid profile of cyanide-utilizing Yersinia species

A cyanide-utilizing Yersinia species was isolated from the cyanide-bearing gold-plating industrial wastewater. Analysis of the fatty acid composition of the organism revealed that it contains large amounts of saturated fatty acids. The unsaturated hydroxy- and cyclopropyl-ring-bearing fatty acids are present in low concentrations. A comparison of the fatty acid composition with other Yersinia species shows that the genus Yersinia appears homogeneous, and that fatty-acid data of Yersiniae do not reflect the distance between Yersiniae species.     

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.     

Role of metals in the biological activity of Clostridium botulinum neurotoxins

Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity. While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane. Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity. Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic. We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane.     

Can urine lamivudine be used to monitor antiretroviral treatment adherence?

Patient adherence to treatment is an important factor in the effectiveness of antiretroviral regimens. Adherence to treatment could be monitored by estimation of antiretroviral drugs in biological fluids. We aimed to obtain information on the quantity and duration of excretion of lamivudine in urine following oral administration of a single dose of 300 mg and to assess its suitability for adherence monitoring purposes. Spot urine samples were collected before dosing and at 4, 8, 12, 24, 28, 32, 48, 72, and 96 hours post dosing from 10 healthy subjects, and lamivudine was estimated by high-pressure liquid chromatography (HPLC). Lamivudine values were expressed as a ratio of urine creatinine. About 91% of the ingested drug was excreted by 24 hours, and the concentration thereafter in urine was very negligible. A lamivudine value of 0.035 mg/mg creatinine or less at 48 hours is suggestive of a missed dose in the last 24 hours. The study findings showed that estimation of urine lamivudine in spot specimens could be useful in monitoring patient adherence to antiretroviral treatment. However, this needs to be confirmed on a larger sample size and among patients on once-daily and twice-daily treatment regimens.     

Low pH-Induced Pore Formation by the T Domain of Botulinum Toxin Type A is Dependent upon NaCl Concentration

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.