The small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP8 DPP9, leading to allosteric activation of the peptidase. SUMO1-EIL noncompetitive Inhibitor of DPP8 and DPP9 - E67 interacting loop (EIL)
Soaking of macromolecular crystals allows the formation of complexes via diffusion of molecules into a preformed crystal for structural analysis. Soaking offers various advantages over co-crystallization, e.g. small samples and high-throughput experimentation. However, this method has disadvantages, such as inducing mechanical stress on crystals and reduced success rate caused by low affinity/solubility of the ligand. To bypass these issues, the Picodropper was previously developed in the authors' laboratory. This technique aimed to deliver small volumes of compound solution in response to crystal dehydration supported by the Free Mounting System humidity control or by IR-laser-induced protein crystal transformation. Herein, a new related soaking development, the Aerosol-Generator, is introduced. This device delivers compounds onto the solution-free surface of protein crystals using an ultrasonic technique. The produced aerosol stream enables an easier and more accurate control of solution volumes, reduced crystal handling, and crystal-size-independent soaking. The Aerosol-Generator has been used to produce complexes of DPP8 crystals, where otherwise regular soaking did not achieve complex formation. These results demonstrate the potential of this device in challenging ligand-binding scenarios and contribute to further understanding of DPP8 inhibitor binding.
        
Title: The SUMO1-E67 Interacting Loop Peptide Is an Allosteric Inhibitor of the Dipeptidyl Peptidases 8 and 9 Pilla E, Kilisch M, Lenz C, Urlaub H, Geiss-Friedlander R Ref: Journal of Biological Chemistry, 288:32787, 2013 : PubMed
The intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nm) and DPP9 (170 nm) over DPPIV (200 mum). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity.