Probing the interaction interface of the GADD45 beta/MKK7 and MKK7/DTP3 complexes by chemical cross-linking mass spectrometry

Enzymatic MS foot-printing and diazirine-based chemical cross-linking MS (CX-MS) strategies were applied to study the interactions between GADD45 beta and MKK7 kinase domain (MKK7_KD) and between DTP3 and MKK7_KD. Our data show that the binding between GADD45 beta and MKK7 largely occurs between GADD45 beta loop 2 (region 103-117) and the kinase enzymatic pocket. We also show that DTP3 interferes with this GADD45 beta/MKK7 interaction by contacting the MKK7 peptides, 113-136 and 259-274. Accordingly, an MKK7_KD Delta(101-136) variant lacking Trpl 35 did not produce a fluorescence quenching effect upon the binding of DTP3. The assessment of the interaction between GADD45 beta 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 GADD45 beta/MKK7 interaction by DTP3 and pave the way to the design of small-molecule DTP3 analogues. (C) 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license.

GADD45 beta is selectively and constitutively expressed in Multiple Myeloma cells, and this expression correlates with an unfavourable clinical outcome. GADD45 beta 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 GADD45 beta/MKK7 complex and is able to restore MKK7/JNK activation, thereby promoting selective cell death of GADD45 beta-overexpressing cancer cells.