Structural and biochemical characterization of the KLHL3WNK 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 (CRL3 KLHL3 ) 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 CRL3 KLHL3 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 -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.

..., College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K. Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K. 1 These authors contributed equally to this work. 2 Correspondence may be addressed to either of these authors (email alex.bullock@sgc.ox.ac.uk or t.kurz@dundee.ac.uk ). The structural co-ordinates reported for KLHL2 and KLHL3 will appear in the PDB under codes 4CHB and 4CH9 respectively. 18 3 2014 13 5

... WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain -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

... the interaction between KLHL3 and WNK, identifying the WNK degron, and present the crystal structure of the KLHL3WNK degron complex. Bric-a-brac Tramtrack and Broad complex (BTB domain) Cullin hypertension Kelch-like protein (KLHL) Kelch-like protein 2 (KLHL2) ubiquitin BTB, Bric-a-brac, Tramtrack, and Broad complex CRL3 KLHL3 , Cullin3-RING ligase in complex with KLHL CUL3, Cullin3 KEAP1, Kelch-like enoyl-CoA hydratase-associated protein 1 KLHL, Kelch-like protein NCC, Na + /Cl ion co

... lower-affinity degron-binding site. Both sites need to be engaged by two Kelch domains of dimeric KEAP1 to appropriately orient NRF2 for ubiquitylation [ 24 ]. Another member of the BTB-Kelch family of proteins is KLHL2 (also known as Mayven). KLHL2 shares 84% sequence identity with KLHL3 across the Kelch domain, and 86% identity overall (see Figure 2 A). KLHL2 has also been implicated recently as a CUL3 substrate adaptor for WNK kinases [ 25 ]. In the present study, we scrutinize the interaction between the WNK degron motif and KLHL3 as well as KLHL2. The data reveal the fundamental features of this interaction and explain how several of the disease-causing mutations disrupt binding. Our results also reveal that the WNK degron motif binds to KLHL3in a markedly different manner to how NRF2 binds to KEAP1, which is the only other BTB-Kelch protein for which substrate interactions have been analysed at the structural level thus far. MATERIALS AND METHODS Materials Lumio Green, Colloidal Blue

... ] with a TEV (tobacco etch virus) protease site downstream of the DAC tag. DNA sequencing was performed by The Sequencing Service, College of Life Sciences, University of Dundee, U.K. ( www.dnaseq.co.uk ). For structure determination, human KLHL2 (IMAGE clone 4791972; residues 294593) and human KLHL3 (NM_017415.2; residues 298587) were cloned into the expression vector pNIC28-Bsa4 (GenBank accession number EF198106) by ligation-independent cloning. This vector encodes for an N-terminal His 6 tag and

... disassociation constant and associated S.E.M. was obtained. All experimental bindings were repeated a minimum of two times and comparable results to those shown in the present study were obtained. Protein expression and purification for crystallization For structure determination, human KLHL2 (residues 294593) and KLHL3 (residues 298587) were expressed from the vector pNIC28-Bsa4in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1litre) in Terrific broth were incubated

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Gene: KLHL2 - kelch-like family member 2 - (NCBI)

Protein : 4chb

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