Self-adaptive Scouting---Autonomous Experimentation for Systems Biology

We introduce a new algorithm for autonomous experimentation. This algorithm uses evolution to drive exploration during scientific discovery. Population size and mutation strength are self-adaptive. The only variables remaining to be set are the limits and maximum resolution of the parameters in the experiment. In practice, these are determined by instrumentation. Aside from conducting physical experiments, the algorithm is a valuable tool for investigating simulation models of biological systems. We illustrate the operation of the algorithm on a model of HIV-immune system interaction. Finally, the difference between scouting and optimization is discussed

The Journal of Biological Chemistry / Structural analyses of Arabidopsis thaliana legumain reveal differential recognition and processing of proteolysis and ligation substrates

Legumain is a dual-function proteasepeptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana legumain isoform (AtLEG) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEG structures disclosed a substrate-induced disorderorder transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEG ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substratelike binding to the active sitebinding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2′S2′, to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEG active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.W_01213M1901(VLID)266778

ACS Catalysis / Distinct roles of catalytic cysteine and histidine in the protease and ligase mechanisms of human legumain as revealed by DFT-based QM/MM simulations

The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [Baker, E. N. J. Mol. Biol. 1980, 141, 441484; Baker, E. N.; J. Mol. Biol. 1977, 111, 207210; Drenth, J.; Biochemistry 1976, 15, 37313738; Menard, R.; J. Cell. Biochem. 1994, 137; Polgar, L. Eur. J. Biochem. 1978, 88, 513521; Storer, A. C.; Methods Enzymol. 1994, 244, 486500. Remarkably, legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given legumains important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields.(VLID)223311