Two-dimensional strandness-dependent electrophoresis

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.     

Structural insights into selectivity and cofactor binding in snake venom L-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH₃) and hydrogen peroxide (H₂O₂). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1 Å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62–71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode.     

Crystal structure of Jararacussin‐I: The highly negatively charged catalytic interface contributes to macromolecular selectivity in snake venom thrombin‐like enzymes

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin‐like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin‐I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three‐dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin‐I is highly negatively charged, which contributes to its unique macromolecular selectivity.     

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.     

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.     

Crystal structure of a dimeric Ser49 PLA₂-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA?-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined. The toxin is devoid of phospholipolytic activity. The structure demonstrates a formation of dimers. In the dimers functionally important peptide segments, located on the protein surface, point in the same direction which can strengthen the pharmacological effect. This supports the hypothesis about the physiological importance of the toxin oligomerization for the myotoxicity and membrane damage. The crystallographic model revealed that the structural determinants of myotoxicity (a positively charged C-terminal region and a hydrophobic knuckle) are fully exposed on the protein surface and accessible for interactions with target membranes. Distortion of the catalytic site region explains the absence of enzymatic activity. The structure reveals anion-binding sites which can be considered as possible sites of interactions of the toxin with a negatively charged membrane surface. The high structural similarity of the Ser49 myotoxin and Asp49 PLA? from the same venom suggests an evolutionary relationship: probably, the Ser49 myotoxin is a product of evolution of the catalytically active phospholipase A?. The first toxin lost the enzymatic activity which is not necessary for the myotoxicity but preserved the cytotoxicity and membrane damaging activity as important components of the venom toxicity.     

Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.     

Crystal structure of myotoxin II, a monomeric Lys49-phospholipase A2 homologue isolated from the venom of Cerrophidion (Bothrops) godmani

Lys49-Phospholipase A2 (Lys49-PLA2) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. With the aim of determining the structural basis for this novel activity, we have solved the crystal structure of myotoxin-II, a Lys49-PLA2 isolated from the venom of Cerrophidion (Bothrops) godmani (godMT-II) at 2.8 A resolution by molecular replacement. The final model has been refined to a final crystallografic residual (Rfactor) of 18.8% (Rfree = 28.2%), with excellent stereochemistry. godMT-II is also monomeric in the crystalline state, and small-angle X-ray scattering results demonstrate that the protein is monomeric in solution under fisicochemical conditions similar to those used in the crystallographic studies.     

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.