Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: In addition to an N-terminal kinase domain, WNK1-4 contain an acidic degron motif that mediates their recruitment to the KLHL3 E3 ligase. Degron sequences are shown for each family member. The WNK3 degron motif contains four amino acid substitutions highlighted with bold underlined letters. Coil indicates a C-terminal coiled-coil domain. CCT-like is a circular permutant of the CCT domain of the OSR1 and SPAK kinases. WNK kinases contain additional RFXV or RFXI motifs (where X denotes any amino acid) that mediate their recruitment to the CCT domain of SPAK/OSR1.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: Purified GST-KLHL3 a.a. 298–587(end) was diluted appropriately and mixed at a 1:1 volume ratio with 20 nM Lumino-Green-labelled WNK3 peptides to the concentration stated in the Figure, with the peptide concentration consistent at 10 nM. Fluorescence polarisation measurements were recorded and corrected to the fluorescent probe alone. Each data point represents three technical replicates. One site-specific binding with hill slope was assumed and the disassociation constant was obtained. Binding curves, assuming one-site-specific binding, were then generated with Prism6 using milli-polarization (mP) units.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques: Purification, Concentration Assay, Fluorescence, Binding Assay, Generated

Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: Overview of the structure of the KLHL3 Kelch domain (rainbow ribbon) in complex with WNK3 peptide (purple sticks). Kelch repeats forming blades I to VI are labelled. N and C termini for both KLHL3 and WNK3 are labelled.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: Stick representation and 2Fo-Fc electron density maps contoured at 1.0 σ for ( A ) WNK3 chain C and ( B ) WNK3 chain D. WNK3 residues and their numbers are labelled (PDB 5NKP). ( C ) Stick representation of WNK4 degron peptide from PDB 4CH9. Residues are labelled as shown. ( D ) Superposition of the KLHL3-WNK3 and KLHL4-WNK4 complexes (PDBs 5NKP and 4CH9, respectively). The Kelch domains of KLHL3 are illustrated in ribbon representation. KLHL3 residues that adopt different conformations are highlighted with stick representation.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: Overview of the polar contacts in the complex interface. KLHL3 residues are labelled in purple and WNK3 peptide is shown in yellow. Hydrogen bonds are demonstrated using dashed lines. Inset panels show selected contacts. Hydrogen bond distances (Å) are indicated.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2020.06.29.178285

Figure Lengend Snippet: ( A ) Location of WNK4 Pro561 at the centre of the KLHL3-WNK4 binding interface (PDB 4CH9). KLHL3 is shown in light purple and WNK4 is shown in light blue. ( B ) Similar position of WNK3 Thr541 in the structure of the KLHL3-WNK3 complex (PDB 5NKP). WNK3 is shown in yellow. ( C ) The modelling of a phosphothreonine in the crystallized conformation of WNK3 Thr541 reveals a severe steric clash with WNK3 Asp544 that would break the critical salt bridge (yellow dashed lines) between this aspartate and KLHL3 Arg528. ( D ) Alternative binding pose of WNK3 Thr541 suggested after an energy minimization procedure to explore other potential side chain conformations. In this alternative pose, the phosphate moiety of pThr541 packs between KLHL3 Arg339 and Tyr577 in a sterically crowded environment that would bury the negative charge unfavourably without hydrogen bonding. ( E ) The modelling of a WNK4 PHAII mutation P561L reveals a steric clash (broken lines) with KLHL3 Arg339 that would disrupt the KLHL3-WNK4 interaction. (F) The modelling of Akt and PKA-dependent phosphorylation of KLHL3 Ser433 shows a severe steric clash with WNK3 Val543 that impairs KLHL3-WNK3 binding. The modelled residues in panels C-F are shown in pink.

Article Snippet: For structure determination, human KLHL3 (a.a. 298–587) was cloned into the pNIC28-Bsa4 vector (Addgene plasmid #110251), which provides an N-terminal hexahistidine tag as previously described [ ].

Techniques: Binding Assay, Mutagenesis, Phospho-proteomics