Cullin3 - BTB Interface: A Novel Target for Stapled Peptides

Abstract

Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3–BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the “stapling” with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49–68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300–600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.

The BTB domains of the potassium channel tetramerization domain proteins prevalently assume pentameric states

“…The BTB domains of KCTD5 (residues 44–145, KCTD5 BTB ), KCTD6 (residues 10–110, KCTD6 BTB ), KCTD11 (residues 15–116, KCTD11 BTB ), KCTD12 (residues 27–131, KCTD12 BTB ), and KCTD15 (residues 61–157, KCTD15 BTB ) were expressed in Escherichia coli BL21(DE3) and were purified by following the previously reported protocols . Similarly, Cul3 NTD , which corresponds to the N‐terminal region (residues 20–381) of Cul3, was expressed and purified according to the procedure reported by Balasco et al .…”

Functional analyses yield detailed insight into the mechanism of thrombin inhibition by the antihemostatic salivary protein cE5 from Anopheles gambiae

“…CD spectra were recorded at 20°C in the far UV (190 -260 nm) on a Jasco J-710 spectropolarimeter (31). Each spectrum was obtained averaging three scans, subtracting contributions from corresponding blanks and converting the signal to mean residue ellipticity in units of degree ϫ cm 2 ϫ dmol Ϫ1 .…”

Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

“…Nevertheless, these studies led to the identification of two weak specific binders of Odin‐Sam1. On the basis of these results we can envision the generation of more stable helical peptides with increased rigidity . Moreover, the NMR structure of the most promising S13H7 ligand could surely be exploited in docking studies to plan the chemical modifications needed to achieve efficient and selective binding to the Odin‐Sam1 ML interface and consequently block engagement of EphA2‐Sam.…”