Structural dynamics of WrbA protein probed by mass spectrometry

A. Kádek1,2, Z. Kukačka1,2, D. Řeha3, M. Rosůlek2, O. Vaněk2, O. Ettrichová3, P. Man1,2, R. Ettrich3, P. Novák1,2

1Institute of Microbiology CAS, Vídeňská 1083, Prague
2Faculty of Science, Charles University in Prague, Hlavova 8, Prague
3Institute of nanobiology and structural biology CAS, Zámek 1, Nové Hrady

Tryptophan repressor-binding protein A (WrbA), more correctly called FMN-dependent NAD(P)H:quinone oxidoreductase, is a protein highly interesting for its involvement in oxidative stress defense responses in many organisms. Its biologically functional assembly is a homotetramer held together by non-covalent interactions, whose 1.2 Å high‑resolution structure has recently been solved by X-ray crystallography. [1] The present work was aimed at studying the structure-function relationship of WrbA, with main focus on uncovering the dynamics of its behaviour in solution. Specifically, this meant understanding the effects of factors determining its conformational and oligomeric states, where the protein is known to be present in the solution in dynamic equilibrium between monomeric, dimeric and tetrameric species.

Studied protein was produced by overexpression in its natural producer organism E. coli and purified to homogeneity by size exclusion and affinity chromatography steps, before being subjected to studies by analytical ultracentrifugation and advanced structural mass spectrometry techniques. Native mass spectrometry coupled with ion mobility enabled us to gently transfer the whole non-covalent WrbA assembly into the gas phase while preserving its structure and showed a dramatic stabilizing effect of FMN cofactor on the protein in its tetrameric form. It also uncovered a positive interdomain cooperative effect which seems to be involved in the binding of FMN molecules to individual WrbA subunits.

Behaviour of WrbA in solution was also probed by hydrogen / deuterium exchange and chemical cross-linking in combination with mass spectrometry. These techniques enabled a more detailed description and structural localization of conformational changes in the WrbA macromolecule depending on the presence of cofactor and on the varying solution conditions. We specifically studied the effects of solution temperature and the protein concentration, which according to the analytical ultracentrifugation determine the oligomeric state of WrbA by influencing its dimer-tetramer dynamic equilibrium.

In combination with computational modelling and crystallographic data, the combination of complementary structural mass spectrometry techniques provided a novel insight into the behaviour of the WrbA protein in solution and offered an explanation for the mechanism of its oligomerization.

1. I. Kishko, J. Carey, D. Reha, J. Brynda, R Winkler, B. Harish, R. Guerra, O. Ettrichova, Z. Kukacka, O. Sheryemyetyeva, P. Novak, M. Kuty, I. Kuta Smatanova, R. Ettrich, M. Lapkouski Acta Crystallogr. Sect. D Biol. Crystallogr., 69, (2013), 1748.

This work was partly funded by InStruct, part of the European Strategy Forum on Research Infrastructures (ESFRI) and supported by national member subscriptions; Czech Science Foundation (P207/10/1934); European Regional Development Funds (CZ.1.07/2.3.00/20.0055, CZ.1.07/2.3.00/30.0003 and CZ.1.05/1.1.00/02.0109); Institutional Research Concept of the Institute of Microbiology RVO61388971; Research Project of Charles University (UNCE 204025/2012); Grant Agency of Charles University (800413) and joint Czech-US International Research Cooperation (ME09016).