A Eukaryotic-Like Interaction of Soluble Cyanobacterial Sensory Rhodopsin Transducer with DNA Shenlin Wang 1 , So Young Kim 2 , Kwang-Hwan Jung 2 , Vladimir Ladizhansky 1 and Leonid S. Brown 1 ⁎ 1 Department of Physics, University of Guelph, Ontario, Canada N1G 2W1 2 Department of Life Science and Institute of Biological Interfaces, Sogang University, Seoul 121-742, Korea Received 13 April 2011; received in revised form 1 June 2011; accepted 2 June 2011 Available online 12 June 2011 Edited by M. F. Summers Keywords: photosensory transduction; transcription factors; NMR spectroscopy; DNA binding; secondary structure Anabaena sensory rhodopsin is a recently discovered membrane photo- sensor with a unique signal transduction cascade. It interacts with a soluble tetrameric transducer [Anabaena sensory rhodopsin transducer (ASRT)] that can bind to promoter regions of several genes related to the utilization of light energy. Even though the X-ray crystal structure of ASRT is available, the mechanism of its interaction with DNA is still unknown. We used solution NMR to understand the mechanism of the DNA binding. Both X- ray crystal structures and solution NMR data reveal seven β-strands forming a rigid scaffold (β-face) and a flexible, partially disordered α-face, comprised by the C-termini and loops. We found that the conformation of the α-face in solution is very different from that in the crystals. While the C- termini of crystalline ASRT are solvent exposed and either α-helical or disordered, about half of ASRT monomers in solution feature buried C- terminal β-strand, with another half of C-tails being random coils. Titration of ASRT with a 20-bp fragment of the pec operon promoter showed that only monomers with β-structured C-tails bind the DNA. NMR signals suggest that specific Arg and Asn/Gln residues are involved in the interaction with DNA. The DNA binding occurs with micromolar affinity and a 1:1 stoichiometry (DNA:ASRT tetramer) and results in a significant ordering of the α-face involving the extension of the C-terminal β-strand and reorganization of the first loop. Such induced-fit type of interaction, which mainly utilizes loops between β-strands and results in the increase in their order, is typical for eukaryotic transcription factors of the immuno- globulin-like fold. © 2011 Elsevier Ltd. All rights reserved. Introduction Anabaena sensory rhodopsin (ASR) is the first photosensory rhodopsin discovered in eubacterial domain, in the genome of freshwater cyanobacte- rium Anabaena sp. PCC 7120. 1 Similar to other rhodopsins, ASR is a membrane-embedded protein, composed of seven transmembrane helices, with covalently attached retinal chromophore. 2,3 Unlike in its archaeal homologs, the cytoplasmic half of ASR is unusually hydrophilic and contains several water molecules forming a hydrogen-bonded net- work with polar side chains, which may be related *Corresponding author. E-mail address: lebrown@uoguelph.ca. Abbreviations used: ASR, sensory rhodopsin from Anabaena (Nostoc) sp. PCC 7120; ASRT, Anabaena sensory rhodopsin transducer; CSI, chemical shift index; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser enhancement spectroscopy; TROSY, transverse relaxation optimized spectroscopy; PDB, Protein Data Bank; NOE, nuclear Overhauser enhancement; dsDNA, double-stranded DNA. doi:10.1016/j.jmb.2011.06.002 J. Mol. Biol. (2011) 411, 449–462 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.