Mutation studies and structure-based identification of potential inhibitor molecules against periplasmic amino acid binding protein of Candidatus Liberibacter asiaticus (CLasTcyA)

Earlier reported crystal structure of CLasTcyA revealed unique features like relatively a larger substrate binding pocket, an extended C-terminal loop restricted by a disulfide bond and involvement of residues from hinge region in substrate binding. In present study, CLasTcyA mutants were created to evaluate the importance of these unique features through biophysical characterization. The Val58 in CLasTcyA was replaced by Trp, conserved in most cystine binding proteins, to reduce the size of the binding pocket. All other mutations were created in CLasTcyA(V58W) mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyA(V58W) showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyA(V58W/C2125/C239S), removal of C-terminal extended loop in truncated CLasTcyA(V58W/C2125) and mutation of His95 from hinge region in the double mutant CLasTcyA(V58W/H95A) showed a marked decrease in stability-indicating the importance of the unique features in structure of CLasTcyA.The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules for detailed future studies. The results clearly establish the importance of unique features in structure-function relationship of CLasTcyA. (C) 2019 Elsevier B.V. All rights reserved.