1. Probing the interaction interface of the GADD45β/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry
- Author
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Emanuela Iaccarino, Rosita Russo, Laura Tornatore, Domenico Raimondo, Angela Chambery, Edoardo Milanetti, Annamaria Sandomenico, Annalia Focà, Paolo V. Pedone, Camilla Rega, Menotti Ruvo, Guido Franzoso, Rega, Camilla, Russo, Rosita, Focà, Annalia, Sandomenico, Annamaria, Iaccarino, Emanuela, Raimondo, Domenico, Milanetti, Edoardo, Tornatore, Laura, Franzoso, Guido, Pedone, Paolo Vincenzo, Ruvo, Menotti, Chambery, Angela, Medical Research Council (MRC), and Cancer Research UK
- Subjects
0301 basic medicine ,Programmed cell death ,Polymers ,MKK7 ,MAP Kinase Kinase 7 ,Mass spectrometry ,Biochemistry ,Article ,Protein–protein interaction ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein-protein interaction ,Structural Biology ,GADD45β, MKK7, Protein-protein interaction, Chemical cross-linking, Mass spectrometry ,Humans ,Protein Kinase Inhibitors ,Molecular Biology ,chemistry.chemical_classification ,Kinase ,0601 Biochemistry And Cell Biology ,General Medicine ,Antigens, Differentiation ,030104 developmental biology ,Enzyme ,chemistry ,Protein kinase domain ,Multiprotein Complexes ,030220 oncology & carcinogenesis ,Chemical cross-linking ,Diazirine ,Cancer cell ,Biophysics ,GADD45β ,Peptides - Abstract
GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Δ(101-136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues. GADD45β is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45β physically interacts with the JNK kinase, MKK7, inhibiting its activity to enable the survival of cancer cells. DTP3 is a small peptide inhibitor of the GADD45β/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45β-overexpressing cancer cells. Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45β and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45β and MKK7 largely occurs between GADD45β loop 2 (region 103–117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45β/MKK7 interaction by contacting the MKK7 peptides, 113–136 and 259–274. Accordingly, an MKK7_KD Δ(101–136) variant lacking Trp135 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45β and MKK7 and the elucidation of the recognition surfaces between DTP3 and MKK7 significantly advance the understanding of the mechanism underlying the inhibition of the GADD45β/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues.
- Published
- 2018