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Maharani Pertiwi Koentjoro

Maharani Pertiwi Koentjoro

Gifu University, Japan

Title: Crystal structure of DNA-Binding Domain-CbnR with its promoter reveals the basis of the LysR-Type Transcriptional Regulator recognition

Biography

Biography: Maharani Pertiwi Koentjoro

Abstract

Cupriavidus necator NH9, which can utilize chlorocatechol as a sole carbon and energy source, degrades chlorocatechol with enzymes of the ortho-cleavage pathway. These enzymes are coded in the cbnABCD operon, of which expression is specifically regulated by a LysR-type transcriptional regulator (LTTR) CbnR. CbnR forms a tetramer and can be regarded as a dimer of dimers. The tetrameric CbnR has four DNA- binding domains and these DNA-binding domains recognize approximately 60 bp DNA sequence. The binding sequence is composed of two binding sites, recognition binding site (RBS)  and  activation  binding site (ABS). Each binding site seems to be recognized by two DNA-binding domains in the tetramer. While the crystal structure of the tetrameric CbnR has already been determined1, the molecular mechanism of DNA recognition by CbnR remains elusive. We therefore initiated the crystal structure analysis of DNA-binding domain of CbnR (CbnR(DBD))    in complex with RBS. The crystal structure would give an insight into the molecular mechanism of the CbnR-DNA interaction, which is the first step to understand the gene activation mechanism by LTTR.

Here we report the crystal structure of CbnR(DBD) (residues 1 - 87) in complex with RBS, a 25-bp DNA fragment. The crystal structure was determined by the MR-native SAD method at 2.55 Å resolution with Rwork/Rfree of 0.221/0.264. The crystal structure shows that dimeric CbnR(DBD) interacts with RBS. The dimeric CbnR(DBD) adopts essentially the same conformation as that in the tetramic CbnR with the root mean squares deviation of 1.1 Å (174 Cα atoms). The a3 helix and the winged region of the winged-helix turn helix (wHTH) motif in CbnR(DBD) directly interact with the major and minor grooves of promoter sequence, respectively, and the interactions seem to bend DNA by approximately 30°. To further analyse the molecular mechanism of their interaction, biochemical analysis is in progress.

References:

  1. Muraoka, S., Okumura, R.., Uragami, Y., Nonaka, T., Ogawa, N., Miyashita, K. and Senda, T (2003) Purification and crystallization of a Lysr–type transcriptional regulator CbnR from Ralstonia eutropha NH9. Protein and Peptide Letters, 10 (3): 325–329.

  2. Ogawa, N. McFall, SM., Klem, TJ., Miyashita, K and Chakrabarty, AM (1999) Transcriptional activation of the clorocatechol degradative genes of Ralstonia eutropha NH9. Journal of Bacteriology, 181 (21): 6697–6705.

  3. Ruangprasert, A., Craven, SH., Neidle, EL., and Momany, C. 2010. Full–length structures of BenM and two variants reveal different oligomerization schemes for LysR–Type transcriptional regulators. Journal of Molecular Biology, 404:568–586.

  4. Devese, L., Smirnova, I., Lonneborg, R., Kapp, U., Brzezinski, P., Leonard, GA. and Dian, C (2011) Crystal structures of  DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR–type transcriptional regulators. Molecular Microbiology, 81 (2): 354–367.

  5. Alanazi, AM., Neidle, EL., and Momany, C (2013) The DNA-binding domain of BenM reveals the structural basis for the recognition   of a T-N11-A sequence motif by LysR-type transcriptional regulators. Acta Crystallographica, D69: 1995-2007.