Intracellular Neutralization of Ricin Toxin by Single-domain Antibodies Targeting the Active Site.

The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near-perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (VHs) previously reported to recognize epitopes in proximity to RTA's active site. Three of the VHs, V2A11, V8E6, and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies ("intrabodies").

A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site.

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot ("cluster 3") located in close proximity to RTA's active site. HX-MS analysis revealed that the 21 VHs recognized four distinct epitope subclusters (3.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of Rv3168 from Mycobacterium tuberculosis H37Rv.

Tuberculosis is a widespread and deadly infectious disease, with one third of the human population already being infected. Aminoglycoside antibiotics have become less effective in recent years owing to antibiotic resistance, which arises primarily through enzymatic modification of the antibiotics. The gene product Rv3168, a putative aminoglycoside phosphotransferase (APH), from Mycobacterium tuberculosis was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.

Crystallization and preliminary X-ray studies of TON_0559, a putative member of the haloacid dehalogenase (HAD) superfamily from Thermococcus onnurineus NA1.

To elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism of the enzymes belonging to the haloacid dehalogenase (HAD) superfamily, TON_0559, a putative HAD subfamily protein from a hyperthermophilic archaeon Thermococcus onnurineus NA1, was expressed, purified and crystallized. X-ray diffraction data were collected to 2.0 A resolution.

The crystal structure of guamerin in complex with chymotrypsin and the development of an elastase-specific inhibitor.

Guamerin, a canonical serine protease inhibitor from Hirudo nipponia, was identified as an elastase-specific inhibitor and has potential application in various diseases caused by elevated elastase concentration. However, the application of guamerin is limited because it also shows inhibitory activity against other proteases. To improve the selectivity of guamerin as an elastase inhibitor, it is essential to understand the binding mode of the inhibitor to elastase and to other proteases.