title: Structural and biochemical characterization of the KLHL3-WNK kinase interaction important in blood pressure regulation.
abstract: WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Frances-Rose
authors.initials: FR
authors.lastname: Schumacher
authors.affiliation: †Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Sorrell
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
authors.affiliation: †Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.
authors.forename: Alex N
authors.initials: AN
authors.lastname: Bullock
authors.affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Thimo
authors.initials: T
authors.lastname: Kurz
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D001794:Blood Pressure / Q000235:genetics / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D044842:Cullin Proteins / Q000235:genetics / Q000502:physiology; D006801:Humans; D006973:Hypertension / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D008840:Microfilament Proteins / Q000502:physiology; D015778:Minor Histocompatibility Antigens; D009419:Nerve Tissue Proteins / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D054875:Ubiquitination; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C485646:KLHL2 protein, human; C489837:KLHL3 protein, human; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D009419:Nerve Tissue Proteins; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; C433806:WNK4 protein, human