Crosslinking studies of protein-protein interactions in nonribosomal peptide biosynthesis.
Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Gene H
GH
Hur
Jordan L
JL
Meier
Jeremy
J
Baskin
Julian A
JA
Codelli
Carolyn R
CR
Bertozzi
Mohamed A
MA
Marahiel
Michael D
MD
Burkart
D000480:Alkynes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D003300:Copper / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D010204:Pantetheine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D046908:Peptide Biosynthesis, Nucleic Acid-Independent; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*
D000480:Alkynes; D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010204:Pantetheine; D003300:Copper; D010453:Peptide Synthases