Molecular dynamics comparison of E. coli WrbA apoprotein and holoprotein

David Řeha¹, Balasubramanian Harish³, Dhiraj Sinha¹, Zdeněk Kukačka², James McSally³, Olga Ettrichová¹, Petr Novák², Jannette Carey³, Rüdiger Ettrich¹

¹Institute of Nanobiology and Structural Biology, Global Change Research Center, Academy of Sciences of the Czech Republic, Zamek 136, 373 33 Nove Hrady, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic,

Vídeňská 1083, 142 20 Praha 4, Czech Republic

³Princeton University, Princeton, New Jersey 08544-1009, USA

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 sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ~5 hours and resulting in complete conversion at the active-site methionine with partial conversion at second, heterogeneous sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidate 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. Furthermore the MD results hint the possibility of communication between binding sites suggesting that FMN binding to WrbA might be cooperative. Indeed, a ready mechanism for allosteric signal transmission exists in the fact that each FMN binding site comprises residues from three of the four WrbA subunits. However there are no conclusive experimantal results so far.

We gratefully acknowledge support from the Czech Science Foundation (P207/10/1934) and joint Czech - US National Science Foundation International Research Cooperation (OISE08-53423).