Structural organization of WrbA in apo- and holoprotein crystals ☆ Julie Wolfova a,b , Ivana Kuta Smatanova a,b , Jiri Brynda c , Jeroen R. Mesters d , Mikalai Lapkouski a,b , Michal Kuty a,b , Antonino Natalello e , Neal Chatterjee f , Sy-Yeu Chern f , Erin Ebbel f , Angela Ricci f , Rita Grandori e , Rüdiger Ettrich a,b, ⁎, Jannette Carey f, ⁎ a Institute of Physical Biology, University of South Bohemia, Zamek 136, 37333 Nove Hrady, Czech Republic b Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zamek 136, 37333 Nove Hrady, Czech Republic c Institute of Molecular Genetics and Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, CZ-166 10 Prague 6, Czech Republic d Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany e Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy f Department of Chemistry, Princeton University, Princeton, NJ 08544-1009, USA abstract article info Article history: Received 16 June 2009 Received in revised form 13 July 2009 Accepted 15 July 2009 Available online 7 August 2009 Keywords: Twisted open-sheet fold Electrostatic potential surface Dimerization Trichloroacetic acid Disulfide NAD(P)H:quinone oxidoreductase Diffraction resolution Two previously reported holoprotein crystal forms of the flavodoxin-like E. coli protein WrbA, diffracting to 2.6 and 2.0 Å resolution, and new crystals of WrbA apoprotein diffracting to 1.85 Å, are refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the protein's global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features. © 2009 Published by Elsevier B.V. 1. Introduction WrbAs 1 are widely distributed novel flavodoxin-like proteins implicated in defense against oxidative stress [1], consistent with their NAD(P)H:quinone oxidoreductase (Nqo) activity [2]. WrbAs appear to bridge bacterial flavodoxins (Fxns) and eukaryotic Nqos structurally and biochemically [3], using FMN as cofactor like the monomeric flavodoxins that transfer single electrons, but forming multimers and carrying out two-electron reduction like the FAD- dependent Nqos [4,5]. WrbAs form homotetramers with intersubunit contacts thought to be mediated by characteristic short sequence insertions that form additional secondary structure elements sur- rounding the twisted open-sheet flavodoxin fold [3,6,7]; E. coli WrbA undergoes a monomer–dimer–tetramer assembly equilibrium in solution [8]. FMN binding is accompanied by a global reduction in protein dynamics, and shifts the subunit assembly equilibrium toward the tetrameric form without altering the monomer–dimer equilibri- um [9]. Residues from three of the four subunits contribute to each FMN-binding site, indicating probable function of WrbAs as obligate tetramers using binding-site residues that are partly conserved in monomeric flavodoxins. The many similarities and distinctions between WrbAs and Fxns extend to their physiological roles, ironically because those of WrbAs are largely unknown and those of Fxns are surprisingly unclear considering their biochemical and redox properties have been intensively studied for so long. The role of Fxns seems clearest in photosynthesis, where they accept electrons from photosystem I. The usual electron acceptor for photosystem I is ferredoxin, with Fxn acting as an alternative electron acceptor whose expression is induced in iron-poor and other stressed growth conditions. This role echoes the role in stress defense that has become a hallmark of the WrbA family, although for both protein families the precise physiological relevance to the stress response is still unclear. In particular, terrestrial plants lack Fxn [10] despite the paucity of iron in many soil types and the evidence that transgenic plants expressing Fxn in their chloroplasts are protected Biochimica et Biophysica Acta 1794 (2009) 1288–1298 ☆ Atomic coordinates have been deposited in the RCSB Protein Data Bank with accession codes 2R96, 2R97, and 2RG1. ⁎ Corresponding authors. R. Ettrich is to be contacted at Institute of Systems Biology and Ecology, Academy of Sciences, Czech Republic. E-mail addresses: ettrich@nh.usbe.cas.cz (R. Ettrich), jcarey@princeton.edu (J. Carey). 1 Although no physiological basis has yet been discovered to support the etymology of the acronym (tryptophan (W) repressor-binding protein A), wrba is the Czech word for willow and the acronym WrbA can be pronounced ver'bah, plural ver'bahs. 1570-9639/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.bbapap.2009.08.001 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap