ORIGINAL PAPER Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein David Reha & Balasubramanian Harish & Dhiraj Sinha & Zdenek Kukacka & James McSally & Olga Ettrichova & Petr Novak & Jannette Carey & Rüdiger Ettrich Received: 29 December 2013 /Accepted: 23 July 2014 /Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfox- ide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxida- tion states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconsti- tution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the sim- ulation results, the first reported for WrbA, led to no hypoth- eses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly. Keywords Global motions . Force field parametrization . Binding site volume . Electrostatic potential surface . NAD(P)H:quinone oxidoreductase Introduction E. coli protein WrbA ([1]; Fig. 1) is the founding member of a class of novel flavoproteins conserved from bacteria to higher plants [2] whose exact functional role is still unknown [3]. Like the flavodoxins to which it is distantly related, WrbA can use its physiological cofactor FMN [4] to transfer electrons [5], but unlike the flavodoxins WrbA transfers two electrons at a time [6] from NADH to quinones. This function is similar to that of the mammalian FAD-dependent NAD (P) H:quinone oxidoreductases [7, 8] to which WrbA is structurally related, suggesting a role in quinone detoxification and oxidative- stress defense. Unlike both these related proteins, WrbA in solution participates in a dimer-tetramer equilibrium [4]. It has thus been suggested [9, 10] that WrbA represents a structural and functional bridge between the monomeric FMN- dependent bacterial flavodoxins and the dimeric FAD- dependent mammalian NAD (P) H-quinone oxidoreductases. This paper belongs to Topical Collection MIB 2013 (Modeling Interactions in Biomolecules VI) Electronic supplementary material The online version of this article (doi:10.1007/s00894-014-2400-8) contains supplementary material, which is available to authorized users. D. Reha (*) : D. Sinha : O. Ettrichova : R. Ettrich Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic e-mail: reha@nh.cas.cz D. Reha : D. Sinha : R. Ettrich Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, 373 33 Nove Hrady, Czech Republic B. Harish : J. McSally : J. Carey Chemistry Department, Princeton University, Princeton, NJ 08544-1009, USA Z. Kukacka : P. Novak Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic Z. Kukacka : P. Novak Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Praha 2, Czech Republic J Mol Model (2014) 20:2400 DOI 10.1007/s00894-014-2400-8