Skip navigation
Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/20085
Title: Deciphering the enigma of missing DNA binding domain of LacI family transcription factors
Authors: Neetu N.
Katiki M.
Mahto J.K.
Sharma M.
Narayanan A.
Maity S.
Tomar, Shailly
Ambatipudi, Kiran S.
Sharma, Ashwani Kumar
Yernool D.
Kumar, Pravindra R.Manish
Published in: Archives of Biochemistry and Biophysics
Abstract: Catabolite repressor activator (Cra) is a member of the LacI family transcriptional regulator distributed across a wide range of bacteria and regulates the carbon metabolism and virulence gene expression. In numerous studies to crystallize the apo form of the LacI family transcription factor, the N-terminal domain (NTD), which functions as a DNA-binding domain, has been enigmatically missing from the final resolved structures. It was speculated that the NTD is disordered or unstable and gets cleaved during crystallization. Here, we have determined the crystal structure of Cra from Escherichia coli (EcCra). The structure revealed a well-defined electron density for the C-terminal domain (CTD). However, electron density was missing for the first 56 amino acids (NTD). Our data reveal for the first time that EcCra undergoes a spontaneous cleavage at the conserved Asn 50 (N50) site, which separates the N-terminal DNA binding domain from the C-terminal effector molecule binding domain. With the site-directed mutagenesis, we confirm the involvement of residue N50 in the spontaneous cleavage phenomenon. Furthermore, the Isothermal titration calorimetry (ITC) assay of the EcCra-NTD with DNA showed EcCra-NTD is in a functional conformation state and retains its DNA binding activity. © 2021 Elsevier Inc.
Citation: Archives of Biochemistry and Biophysics, 713
URI: https://doi.org/10.1016/j.abb.2021.109060
http://repository.iitr.ac.in/handle/123456789/20085
Issue Date: 2021
Publisher: Academic Press Inc.
Keywords: Catabolite repressor activator
Effector molecule
Gene expression
Isothermal titration calorimetry
Protein crystallization
Site-directed mutagenesis
Spontaneous cleavage
Transcription regulator
X-ray crystallography
ISSN: 39861
Author Scopus IDs: 57218693333
57112522900
57194229801
57216602956
36167154300
57208756395
57203506001
14628506400
57214355701
6507388525
55064809000
Author Affiliations: Neetu, N., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Katiki, M., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Mahto, J.K., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Sharma, M., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Narayanan, A., Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University ParkPA 16802, United States
Maity, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Tomar, S., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Ambatipudi, K., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Sharma, A.K., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Yernool, D., Department of Biological Sciences, Purdue University, West LafayetteIN 47906, United States
Kumar, P., Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand 247667, India
Funding Details: This work was supported by a grant from ICMR ( Indian Council of Medical Research ), India (Project No. ICM/2018/001022 ). Neetu would like to thank CSIR; M. Katiki would like to thank MHRD; Jai Krishna Mahto would like to thank DBT. The authors would also like to thank the Macromolecular Crystallographic Unit (MCU) facility at IIC, IIT Roorkee for X-ray data collection and ITC experiments. We also thank the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for X-ray data collection. Department of Biotechnology, Ministry of Science and Technology, India, DBT; Indian Council of Medical Research, ICMR: ICM/2018/001022; Council of Scientific and Industrial Research, India, CSIR; European Synchrotron Radiation Facility, ESRF; Ministry of Education, India, MoE
Corresponding Author: Kumar, P.; Department of Biotechnology, India; email: pravindra.kumar@bt.iitr.ac.in
Appears in Collections:Journal Publications [BT]

Files in This Item:
There are no files associated with this item.
Show full item record


Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.