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

Dana Felker; Haoming Zhang; Zhiyuan Bo; Miranda Lau; Yoshihiro Morishima; Santiago Schnell; Yoichi Osawa

doi:10.1016/j.bpc.2021.106590

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

Biophys Chem. 2021 Jul:274:106590. doi: 10.1016/j.bpc.2021.106590. Epub 2021 Apr 20.

Authors

Dana Felker¹, Haoming Zhang², Zhiyuan Bo³, Miranda Lau⁴, Yoshihiro Morishima⁵, Santiago Schnell⁶, Yoichi Osawa⁷

Affiliations

¹ Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: dbfelker@umich.edu.
² Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: haom@umich.edu.
³ Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: zybo@umich.edu.
⁴ Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: laumiran@umich.edu.
⁵ Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: ymo@umich.edu.
⁶ Department of Molecular & Integrative Physiology, 7744 MS II, 1137 E. Catherine St., Ann Arbor, MI 48109-5622, USA. Electronic address: schnells@umich.edu.
⁷ Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-5632, USA. Electronic address: osawa@umich.edu.

Abstract

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.

Keywords: BM3; CXL-MS; CYP102A1; Crosslinking; Mass spectrometry; Protein-protein interactions.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / chemistry*
Cross-Linking Reagents / chemistry*
Cytochrome P-450 Enzyme System / chemistry*
Mass Spectrometry
Models, Molecular
NADPH-Ferrihemoprotein Reductase / chemistry*
Protein Binding

Substances

Bacterial Proteins
Cross-Linking Reagents
Cytochrome P-450 Enzyme System
NADPH-Ferrihemoprotein Reductase
flavocytochrome P450 BM3 monoxygenases

Abstract

Publication types

MeSH terms

Substances

Grants and funding