Chemical cross-linking and H/D exchange combined with mass spectrometry: a tool to validate and refine 3-D protein X–ray model

Daniel Rozbesky^1,2, Petr Pompach^1,2, Petr Man^1,2, Karel Bezouska^1,2 and Petr Novak^1,2r.dano@centrum.cz

¹Institute of Microbiology, Academy of Science of the Czech Republic, Vídeňská 1083, Prague 4, Czech Republic

²Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, Czech Republic

Chemical cross-linking and Hydrogen/Deuterium exchange combined with mass spectrometry are powerful tools for elucidating protein conformation in solution. We applied both methods to study structural details of an important activating lymphocyte receptor NKR-P1A and to distinguish between structural characteristics in crystal and physiological conditions. The determination of crystal structure of NKR-P1A evokes questions about the unique flap region containing residues Pro161-Asp187 which is in crystal directed towards symmetrically related molecule. Chemical cross-linking and H/D exchange combined with high resolution mass spectrometry have been employed to investigate the flexibility and conformation of the flap region.

In order to study the flexibility and conformation of the flap region in the NKR-P1A protein, we have prepared recombinant NKR-P1A protein and its mutant NKR-P1A-NF in which the flap region containing residues Pro161-Asp187 was deleted and replaced by two alanines. While the main structure of NKR-P1A obtained by mass spectrometry techniques was consistent with the previously known crystal data, a difference was found in the flap region. In the crystal structure the flap region extends from the compact core region. On the contrary, analysis of the peptic fragments showed decreased local H/D exchange in the NKR-P1A protein in region containing residues 115-131, 138-143 and 190-206 in comparison with NKR-P1A-NF. This indicates a reorganization of the flap region and its association with the compact core region. The mass spectrometry results were further confirmed by the NMR structural analysis.