Structural insights into thrombolytic activity of destabilase from medicinal leech.

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

Mechanisms of membrane protein crystallization in 'bicelles'.

Despite remarkable progress, mainly due to the development of LCP and 'bicelle' crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the "bicelle" crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of "bicelle" MP crystallization and will support rational design of crystallization.

Rhodopsin Channel Activity Can Be Evaluated by Measuring the Photocurrent Voltage Dependence in Planar Bilayer Lipid Membranes.

The studies of the functional properties of retinal-containing proteins often include experiments in model membrane systems, e.g., measurements of electric current through planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one of the membrane surfaces. However, the possibilities of this method have not been fully explored yet. We demonstrated that the voltage dependence of stationary photocurrents for two light-sensitive proteins, bacteriorhodopsin (bR) and channelrhodopsin 2 (ChR2), in the presence of protonophore had very different characteristics. In the case of the bR (proton pump), the photocurrent through the BLM did not change direction when the polarity of the applied voltage was switched. In the case of the photosensitive channel protein ChR2, the photocurrent increased with the increase in voltage and the current polarity changed with the change in the voltage polarity. The protonophore 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB) was more efficient in the maximizing stationary photocurrents. In the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), the amplitude of the measured photocurrents for bR significantly decreased, while in the case of ChR2, the photocurrents virtually disappeared. The difference between the effects of TTFB and CCCP was apparently due to the fact that, in contrast to TTFB, CCCP transfers protons across the liposome membranes with a higher rate than through the decane-containing BLM used as a surface for the proteoliposome adsorption.