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

Publisher: Published by Portland Press on behalf of the Biochemical Society Country of Publication: England NLM ID: 2984726R Publication Model: Print Cited Medium: Internet ISSN: 1470-8728 (Electronic) Linking ISSN: 02646021 NLM ISO Abbreviation: Biochem J Subsets: MEDLINE

Original Publication: London, UK : Published by Portland Press on behalf of the Biochemical Society

Hypertension* ; Ubiquitin-Protein Ligases*; Adaptor Proteins, Signal Transducing/genetics ; Humans ; Microfilament Proteins/genetics ; Phosphorylation ; Proline ; Protein Serine-Threonine Kinases/genetics ; Ubiquitin

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.
(© 2022 The Author(s).)

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MC_UU_00001/7 United Kingdom MRC_ Medical Research Council; MC_UU_00018/1 United Kingdom MRC_ Medical Research Council; MC_UU_12016/2 United Kingdom MRC_ Medical Research Council; MR/X006980/1 United Kingdom MRC_ Medical Research Council

Keywords: crystallography; degron; hypertension; mutation; protein-serine-threonine kinases; ubiquitin ligases

0 (Adaptor Proteins, Signal Transducing)
0 (KLHL3 protein, human)
0 (Microfilament Proteins)
0 (Ubiquitin)
9DLQ4CIU6V (Proline)
EC 2.3.2.27 (Ubiquitin-Protein Ligases)
EC 2.7.1.- (WNK2 protein, human)
EC 2.7.11.1 (Protein Serine-Threonine Kinases)

Date Created: 20220218 Date Completed: 20220505 Latest Revision: 20240313

20240313

PMC9022995

10.1042/BCJ20220019

35179207