Structural Elucidation of Peptide Binding to KLHL-12, a Substrate Specific Adapter Protein in a Cul3-Ring E3 Ligase Complex

KLHL-12 is a substrate specific adapter protein for a Cul3-Ring ligase complex. It is a member of the Kelch β-propeller domain subclass of Cullin-Ring substrate recognition domains. This E3 ubiquitin ligase complex has many activities, including acting as a negative regulator of the Wnt signaling pathway by mediating ubiquitination and subsequent proteolysis of Dvl3/Dsh3. KLHL-12 is also known to mediate the polyubiquitination of the dopamine D4 receptor (D4.2), the ubiquitination of KHSRP, a protein that is involved in IRES translation, and also the ubiquitination of Sec31, which is involved in endoplasmic reticulum–Golgi transport by regulating the size of COPII coats. Earlier studies broadly defined the substrate binding regions for D4.2 and Dvl3/Dsh3 to KLHL-12. We tested several peptides from these regions and succeeded in identifying a short peptide that bound to KLHL-12 with low micromolar affinity. To better understand the sequence specificity of this peptide, we used alanine substitutions to map the important residues and obtained an X-ray structure of this peptide bound to KLHL-12. This structure and our peptide affinity measurements suggest a sequence motif for peptides that bind to the top face of KLHL-12. Understanding this binding site on KLHL-12 may contribute to efforts to find small molecule ligands that can either directly inhibit the degradation of substrate proteins or be used in targeted protein degradation strategies using PROTACs.