In-solution structure of cellobiose dehydrogenase probed by hydrogen / deuterium exchange mass spectrometry

Alan Kádek^1,2, Roland Ludwig³, Petr Halada¹, Petr Man^1,2

¹Institute of Microbiology ASCR, Prague, CZECH REPUBLIC

²Faculty of Science, Charles University in Prague, CZECH REPUBLIC

³University of Natural Resources and Applied Life Sciences, Vienna, AUSTRIA

Cellobiose dehydrogenase (CDH) is an enzyme involved in the early processes of lignocellulose catabolic degradation. Being the only currently known extracellular flavocytochrome, CDH is unique from the point of view of its molecular architecture. It is a monomeric glycoprotein consisting of two domains connected by a flexible linker. The combination of a haem with a FAD molecule within a single protein gives CDH the ability to exchange electrons with a variety of interaction partners, thus making it an interesting enzyme for research in the fields of biocatalysis and biosensors.

To probe the in-solution structural organization of this multidomain enzyme as well as its proposed conformational changes under different pH conditions, we employed amide hydrogen / deuterium exchange in combination with mass spectrometry (HDX-MS).

Prior to HDX-MS experiments we thoroughly characterized primary sequence of CDH using mass spectrometry methods. We examined the disulfide bond organization of CDH, found all six potential N-glycosylation sites in CDH molecule to be decorated with high-mannose type glycans and moreover, we uncovered an extensive O-glycosylation of the interdomain linker region. Next, we optimized digestion conditions and performed HDX-MS experiment examining the conformations at pH 5.4 (enzymatic pH optimum) and pH 7.4 (only residual activity remaining). The data indicate a conformational change near the proposed domain interface, which could be explained by “opening” of the two-domain assembly at higher than optimal pH. These results are in agreement with sedimentation velocity data from analytical ultracentrifugation, which demonstrate a global conformational change in the CDH molecule at pH 5.4 and pH 7.4.

This work has been supported by the Grant Agency of the Czech Republic (GACR P206/12/0503) and by the Institutional Research Project of the Institute of Microbiology (RVO61388971).