Mapping protein-protein interactions in homodimeric CYP102A1 by crosslinking and mass spectrometry


Covalent crosslinking and mass spectrometry techniques hold great potential in the study of multiprotein complexes, but a major challenge is the inability to differentiate intra- and interprotein crosslinks in homomeric complexes. In the current study we use CYP102A1, a wellcharacterized homodimeric P450, to examine a subtractive method that utilizes limited crosslinking with disuccinimidyl dibutyric urea (DSBU) and isolation of the monomer, in addition to the crosslinked dimer, to identify inter-monomer crosslinks. The utility of this approach was examined with the use of MS-cleavable crosslinker DSBU and recently published cryo-EM based structures of the CYP102A1 homodimer. Of the 31 unique crosslinks found, 26 could be fit to the reported structures whereas 5 exceeded the spatial constraints. These suggest increased flexibility of the reductase domain such that the flavins are in closer proximity to the heme