Deciphering the enigma of missing DNA binding domain of LacI family transcription factors

Neetu Neetu, Madhusudhanarao Katiki, Jai Krishna Mahto, Monica Sharma, Anoop Narayanan, Sudipa Maity, Shailly Tomar, Kiran Ambatipudi, Ashwani Kumar Sharma, Dinesh Yernool, Pravindra Kumar

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article number109060
JournalArchives of Biochemistry and Biophysics
Volume713
DOIs
StatePublished - Nov 30 2021

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

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