Crystal Structure of KLHL3 in Complex with Cullin3

Abstract

KLHL3 is a BTB-BACK-Kelch family protein that serves as a substrate adapter in Cullin3 (Cul3) E3 ubiquitin ligase complexes. KLHL3 is highly expressed in distal nephron tubules where it is involved in the regulation of electrolyte homeostasis and blood pressure. Mutations in KLHL3 have been identified in patients with inherited hypertension disorders, and several of the disease-associated mutations are located in the presumed Cul3 binding region. Here, we report the crystal structure of a complex between the KLHL3 BTB-BACK domain dimer and two copies of an N terminal fragment of Cul3. We use isothermal titration calorimetry to directly demonstrate that several of the disease mutations in the KLHL3 BTB-BACK domains disrupt the association with Cul3. Both the BTB and BACK domains contribute to the Cul3 interaction surface, and an extended model of the dimeric CRL3 complex places the two E2 binding sites in a suprafacial arrangement with respect to the presumed substrate-binding sites.

Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase

“…structures of BTB-Cul3 complexes have subsequently been solved (Errington et al 2012;Ji and Prive 2013), and these models confirmed that our mutagenesis approach targeted residues that contribute to the Cul3-BTB binding interface (Figure 4, A-B).…”

“…Kelch residues targeted in this study were generally not conserved in SPOP and were modeled into this representation using MacPyMol. The amino acid sequence positions targeted for mutagenesis are oriented toward Cul3, and each was found to contribute to the interaction surface in the solved structures (Errington et al 2012;Ji and Prive 2013). (C) Western analysis of ovaries from kel DE1 females rescued with Kelch containing the indicated mutations in the BTB domain.…”

“…Structural analysis identified a hydrophobic/acidic "F-X-E" amino acid triplet in this loop that is unstructured in crystals of BTB domains alone, but forms a short a-helix in crystals with BTB domains bound to Cul3 (Errington et al 2012;Ji and Prive 2013). The hydrophobic F residue in this motif is buried in a nonpolar pocket on Cul3, and the highly conserved glutamic acid residue forms a hydrogen bond with Cul3.…”

“…Two of the six residues make substantial contacts between the BTB domain and Cul3 (Errington et al 2012), and these correspond to S201 and N234 in Kelch QSTN . Q200 and T231 are nearby residues shown to also contribute to Cul3-BTB binding (Errington et al 2012;Ji and Prive 2013). The rescue of the F-actin cytoskeletal phenotypes by Kelch QSTN suggests that it is still capable of targeting its substrate for ubiquitylation.…”

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases

“…Many types of E3 ligases exist, but the Cullin-RING ligases (CRLs) constitute the largest family. CUL3 acts as the scaffolding protein of subfamily CLR3; in other words, it brings together the substrate recognition protein with the rest of the ubiquitination machinery (22,30). CUL3 binds to an adaptor protein of the BTB (Bric-a-brac, Tramtrack, and Broad complex)-containing family, which in turn binds to the substrate protein being ubiquitylated (8).…”