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

Zhuoyao Chen,Monique P C Mulder,Jinwei Zhang,Raphael Heilig,Francesca D'Amico,Roman Fischer,Vincenzo D'Angiolella,Fiona J Sorrell,Adrián R Murillo-De-Ozores,Alex N Bullock,Dario R Alessi,Charlotte E Manning,María Castañeda-Bueno,Gerardo Gamba

doi:10.1042/bcj20220019

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 four 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.

Highlights

WNK kinases play a key role in mammalian blood pressure regulation [1-3]
The SPAK/OSR1 CCT domain is recruited to RFXV/I motifs in the N-terminus of N[K]CC cation-chloride channels, and thereby phosphorylates conserved threonine residues in their cytoplasmic domains to activate channel activity [10, 12]
We report the 2.8 Å structure of the Kelch-like protein 3 (KLHL3) Kelch domain in complex with the WNK3 degron motif, as well as peptide binding assays that characterise the interactions of KLHL3 with variant WNK3 peptides

Summary

Introduction

WNK (with no lysine) kinases play a key role in mammalian blood pressure regulation [1-3]. WNKs, together with their downstream targets SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), regulate cation-chloride channels to achieve ion homeostasis in the kidney and neurons through a cascade of phosphorylation [3]. Activated WNK kinases stimulate the kinase activity of SPAK/OSR1 by phosphorylating a conserved threonine residue in their kinase activation segments (SPAK Thr233, OSR1 Thr185) [7-9], which is facilitated by interaction between the SPAK/OSR1 CCT (Conserved C-terminal) domain and WNK RFXV/I peptide motifs [10, 11]. The SPAK/OSR1 CCT domain is recruited to RFXV/I motifs in the N-terminus of N[K]CC cation-chloride channels, and thereby phosphorylates conserved threonine residues in their cytoplasmic domains to activate channel activity [10, 12]. Phosphorylation on KCC cation-chloride channels by the WNK-SPAK/OSR1 axis has an inhibitory function [13]. Excessive activity of the WNK-SPAK/OSR1 cascade is the primary cause of PHAII (pseudohypoaldosteronism type II) hypertension and hyperkalemia syndrome [14]

Methods

Results

Conclusion