Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

Interactions of some commonly used drugs with human α-thrombin.

Adverse side effects of drugs are often caused by the interaction of drug molecules to targets other than the intended ones. In this study, we investigated the off-target interactions of some commercially available drugs with human α-thrombin. The drugs used in the study were selected from Super Drug Database based on the structural similarity to a known thrombin inhibitor argatroban. Interactions of these drugs with thrombin were initially checked by in silico docking studies and then confirmed by thrombin inhibition assay using a fluorescence microplate-based method. Results show that the three commonly used drugs piperacillin (anti-bacterial), azlocillin (anti-bacterial), and metolazone (anti-hypertensive and diuretic) have thrombin inhibitory activity almost similar to that of argatroban. The Ki values of piperacillin, azlocillin, and metolazone with thrombin are .55, .95, and .62 nM, respectively. The IC50 values of piperacillin, azlocillin, and metolazone with thrombin are 1.7, 2.9, and 1.92 nM, respectively. This thrombin inhibitory activity might be a reason for the observed side effects of these drugs related to blood coagulation and other thrombin activities. Furthermore, these compounds (drugs) may be used as anti-coagulants as such or with structural modifications.

In silico characterization of a novel ß-1,3-glucanase gene from Bacillus amyloliquefaciens--a bacterial endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii.

We report the molecular characterization of ß-1,3-glucanase-producing Bacillus amyloliquefaciens-an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the ß-1,3-glucanase gene was found to be 747 bp in length. A homology model of the ß-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target ß-1,3-glucanase protein and the template protein, endo ß-1,3-1,4-glucanase protein (PDB ID: 3o5s), were found to share 94% sequence identity and to have similar secondary and tertiary structures. In the modeled structure, three residues in the active site region of the template-Asn52, Ile157 and Val158-were substituted with Asp, Leu and Ala, respectively. Computer-aided docking studies of the substrate disaccharide (ß-1, 3-glucan) with the target as well as with the template proteins showed that the two protein-substrate complexes were stabilized by three hydrogen bonds and by many van der Waals interactions. Although the binding energies and the number of hydrogen bonds were the same in both complexes, the orientations of the substrate in the active sites of the two proteins were different. These variations might be due to the change in the three amino acids in the active site region of the two proteins. The difference in substrate orientation in the active site could also affect the catalytic potential of the ß-1,3 glucanase enzyme.