Biology subjects

Sorrell, F. J.

Publications and source records attributed to Sorrell, F. J..

3 recordsLinked to original sources

bioRxiv2024

Engineering CRBN for rapid identification of next generation binders

The majority of clinical degrader candidates utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target substrate to the E3 ligase receptor Cereblon (CRBN), however, concomitant neo-substrate degradation by IMiDs often results in severe off-target effects. Further biophysical screening is needed to discover CRBN binders that might overcome these safety concerns, but the previously reported CRBN protein constructs suffer significant limitations that reduce their applicability as tools for biophysical assays making large-scale screening efforts a costly endeavor. This is due to the requirement of CRBN co-expression with DDB1 in insect cells to produce soluble protein that contains all the reported structural features necessary for proper compound interaction. Here, a near full-length human CRBN construct was designed that retains these required features, but for the first time allows the generation of highly homogenous and cost-efficient expression in E.coli, eliminating the need for DDB1 co-expression. We have extensively profiled the new construct against the existing CRBN constructs in binary and ternary degrader complexes, showing it to be the "best of both worlds" in terms of binding characteristics and ease of protein production. We then designed the Enamine CRBN focused IMiD library of 4480-compounds and demonstrated applicability of the new construct to high throughput screening by identifying novel CRBN binders with high potency and ligand efficiency. The new methods described here should serve as valuable tools for the development of the next generation IMiD-based degraders.

biophysics↗

bioRxiv2020

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

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 4 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.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsBTBBric-a-brac, Tramtrack, and Broad complexCCTConserved C-terminalCRL3Cullin3-dependent RING E3 ligaseDMEMDulbecco’s modified Eagle’s mediumDTTDithiothreitolFBSfetal bovine serumGSTGlutathione S-transferaseHCDHigher-energy collisional dissociationHEK293Thuman embryonic kidney 293 cell line expressing the SV40 large T antigenKCCK+/Cl− co-transportersIPTGIsopropyl β-D-1-thiogalactopyranosideKLHL3Kelch-like protein 3LC/MSLiquid chromatography-mass spectrometryN[K]CCNa+/Cl− ion co-transportersNKCC2Na+/K+/2Cl− co-transporter 2OSR1oxidative stress-responsive kinase 1PDBProtein DatabankPHAIIpseudohypoaldosteronism type IIRINGreally interesting new geneSPAKSPS1-related proline/alanine-rich kinaseTCEPtris(2-carboxyethyl)phosphineTEVtobacco etch virusWNKwith no lysine (K) kinaseView Full Text

molecular biology↗

bioRxiv2020

STK32A is a dual-specificity AGC kinase with a preference for acidic substrates

The STK32 kinases are a small subfamily of three uncharacterised serine/threonine kinases from the AGC kinase family whose functional role is so far unknown. Here, we analyse the consensus peptide sequence for STK32A phosphorylation, showing that STK32A is directed towards acidic substrate sequences and exhibits dual-specificity for serine/threonine and tyrosine residues. A crystal structure of STK32A reveals an overall structure typical of the AGC protein kinase family but with significant and unique features including an altered binding mode of the hydrophobic motif to the N-terminal lobe of the kinase domain, and a novel alpha-helix in between the turn motif and the hydrophobic motif. The crystal structure combined with phylogenetic analysis reveals the evolutionary conservation of the acidic substrate preference. In vitro binding assays demonstrated that the STK32 kinases bind significant numbers of clinically used kinase inhibitors.

biochemistry↗