Co-crystallization of WrbA protein with its flavin cofactor leads to 3D-structure determination
J. Wolfová1,2, J. Brynda1,3 and I. Kutá Smatanová1,2
of Physical Biology, University of South Bohemia České Budějovice, Zámek 136,
CZ-373 33 Nové Hrady, Czech Republic
2Institute of Systems Biology and Ecology, Academy of Science of the Czech Republic, Zámek 136,CZ-373 33 Nové Hrady, Czech Republic
3Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-16637 Prague 6, Czech Republic
The WrbA protein from Escherichia coli belongs to a new family of multimeric flavoproteins [1,2], that contain flavin mononucleotide (FMN) as a physiological cofactor. The proper function of WrbA had been unknown until recently. According to the latest observations the E. coli WrbA and its homologues exhibit the NAD(P)H:quinone oxidoreductase activity, which is proposed to be implicated in protection mechanisms against oxidative stress . Determination of crystal structure of the E. coli WrbA is aimed at proper characterization of the new protein family with respect to its recently revealed oxidoreductase activity.
Recombinant WrbA protein was expressed in E. coli CY15071(lDE3) cells and purified as described previously . Due to the loss of FMN cofactor during purification, the WrbA apoprotein was crystallized first. Single crystals for diffraction analysis were obtained after optimization of crystallization by using additives (Cd-chloride and Li-citrate) , but due to inconvenient diffraction parameters the structure couldn’t be solved. Crystallizability of the WrbA protein improved significantly after binding of its cofactor, FMN. Yellow, well-formed crystals of WrbA protein in complex with its flavin cofactor were obtained even from several crystallization conditions. Yielding of diffraction-quality WrbA holoprotein crystals without additional optimzation steps shows the positive influence of FMN cofactor on crystallization of WrbA protein. This effect reflects FMN as a potential stabilizer of the protein structure. In correspondence to this finding, FMN binding was shown to stabilize WrbA protein providing it with increased resistance against proteolytic digestion (data not shown) and thermal denaturation . Data collected for the WrbA holoprotein crystals by using synchrotron radiation were used for 3D-structure determination. The structure of WrbA in complex with FMN was refined to 2 Ǻ.
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This work is supported by the Ministry of Education of the Czech Republic (projects: Kontakt ME640, MSM6007665808, LC06010) and by the Academy of Sciences of the Czech Republic (AV0Z60870520). We are grateful to the staff of EMBL/DESY in Hamburg for the support during data collection. Diffraction measurements at the synchrotron DESY/EMBL were supported of the European Community, Research Infrastructure Action under the FP6 “Structuring the European Research Area Specific Programme” to the EMBL Hamburg Outstation, Contract Number RII3-CT-2004-506008.