Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase

Abstract

The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsBTBBric-a-brac, Tramtrack, and Broad complexCCTConserved C-terminalCRL3Cullin3-dependent RING E3 ligaseDMEMDulbecco’s modified Eagle’s mediumDTTDithiothreitolFBSfetal bovine serumGSTGlutathione S-transferaseHCDHigher-energy collisional dissociationHEK293Thuman embryonic kidney 293 cell line expressing the SV40 large T antigenKCCK+/Cl− co-transportersIPTGIsopropyl β-D-1-thiogalactopyranosideKLHL3Kelch-like protein 3LC/MSLiquid chromatography-mass spectrometryN[K]CCNa+/Cl− ion co-transportersNKCC2Na+/K+/2Cl− co-transporter 2OSR1oxidative stress-responsive kinase 1PDBProtein DatabankPHAIIpseudohypoaldosteronism type IIRINGreally interesting new geneSPAKSPS1-related proline/alanine-rich kinaseTCEPtris(2-carboxyethyl)phosphineTEVtobacco etch virusWNKwith no lysine (K) kinaseView Full Text