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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/1164
Title: Mutation studies and structure-based identification of potential inhibitor molecules against periplasmic amino acid binding protein of Candidatus Liberibacter asiaticus (CLasTcyA)
Authors: Kumar P.
Dalal V.
Kokane A.
Singh S.
Lonare S.
Kaur H.
Ghosh D.K.
Kumar P.
Sharma A.K.
Published in: International Journal of Biological Macromolecules
Abstract: 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 CLasTcyAV58W mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyAV58W showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyAV58W/C212S/C239S, removal of C-terminal extended loop in truncated CLasTcyAV58W/C212S and mutation of His95 from hinge region in the double mutant CLasTcyAV58W/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. © 2019 Elsevier B.V.
Citation: International Journal of Biological Macromolecules(2020), 147(): 1228-1238
URI: https://doi.org/10.1016/j.ijbiomac.2019.09.250
http://repository.iitr.ac.in/handle/123456789/1164
Issue Date: 2020
Publisher: Elsevier B.V.
Keywords: ABC transporters
Candidatus Liberibacter asiaticus
Periplasmic amino acid binding protein
Surface plasmon resonance (SPR)
ISSN: 1418130
Author Scopus IDs: 24432768800
57194227044
56074767300
57212373509
57212387983
57212373794
57214787956
55064809000
57214355701
Author Affiliations: Kumar, P., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Dalal, V., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Kokane, A., Plant Virology Laboratory, ICAR- Central Citrus Research Institute, Nagpur, 440 010, India
Singh, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Lonare, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Kaur, H., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Ghosh, D.K., Plant Virology Laboratory, ICAR- Central Citrus Research Institute, Nagpur, 440 010, India
Kumar, P., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Sharma, A.K., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247 667, India
Funding Details: The authors thank the Macromolecular Crystallographic Facility (MCU) at IIC, Indian Institute of Technology Roorkee for using the bioinformatics facility. The authors thank the NMR facility at IIC, IIT Roorkee to carry out fluorescence experiments. We thank Ms. Neetu and Ms. Ruchi for Surface Plasmon Resonance experiments. Kumar. P. thanks UGC, Government of India for providing fellowship. The study was conceived by AKS and DKG. AK have done the genomic DNA isolation in guidance of DKG. PK1, SS, SL and HK designed and performed all experimental work. VD performed bioinformatics studies under supervision of PK2. AKS and PK1 wrote the first draft of manuscript. AKS, PK2 and DKG revised the manuscript. All the authors discussed the results and contributed towards finalizing the manuscript.
Corresponding Author: Sharma, A.K.; Department of Biotechnology, Indian Institute of Technology RoorkeeIndia; email: aksbsfbs@iitr.ac.in
Appears in Collections:Journal Publications [BT]

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