Structural details of changes accompanying interaction between insulin-related hormones and their binding partners are often enigmatic. Here, cross-linking/mass spectrometry could complement structural techniques and reveal details of these protein-protein interfaces. We used such approach to clarify missing structural description of the interface in human insulin-like growth factor (IGF-1): Drosophila melanogaster imaginal morphogenesis protein-late 2 protein (Imp-L2) complex which we studied previously by X-ray crystallography. We crosslinked these proteins by heterobifunctional cross-linker sulfosuccinimidyl 4,4'-azidopentanoate (Sulfo-SDA) for the subsequent mass spectrometry (MS) analysis. The MS analysis revealed IGF-1:Imp-L2 interactions which were not resolved in the crystal structure of this assembly, and they converged with X-ray results, indicating the importance of the IGF-1 N-terminus interaction with the C-terminal (185-242) part of the Imp-L2 for stability of this complex. Here, we also showed the advantage and reliability of MS approach in solving details of protein-protein interactions that are too flexible for solid state structural methods.

Institute of Microbiology Czech Academy of Sciences Prague Czechia

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czechia

York Structural Biology Laboratory Department of Chemistry The University of York York United Kingdom

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