Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: α (26.5 kDa) and β (61.7 kDa). Three (αβ) heterodimers form a trimeric (αβ)₃ assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four (αβ)₃ units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in α, β, and native complex) were assigned. Molecular modeling of urease αβ complex and trimeric urease units (αβ)₃ revealed a linkage site between the α-subunit and the β-subunit, and an internal cross-linkage in the β-subunit.