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 inter- protein crosslinks in homomeric complexes. In the current study we use CYP102A1, a well-characterized 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. Not only did these crosslinks validate the cryo-EM structure, they point to new conformations of CYP102A1 that bring the flavins in closer proximity to the heme.

Automated summary

Covalent crosslinking and mass spectrometry techniques show promise in studying multiprotein complexes, with a study using CYP102A1 homodimer to identify inter-monomer crosslinks through limited crosslinking and monomer isolation. By examining 31 unique crosslinks, validation of cryo-EM structures and new conformations of CYP102A1 were achieved.