# Identification of ZFTA as a Novel KLHL20 Substrate and Mechanistic Insights Into Fuzzy Binding of Disordered Peptides via Biosensor Analysis and Computational Modelling

## TL;DR

Researchers found that the ZFTA protein interacts with KLHL20 through a flexible, weak binding mechanism, suggesting a new role for KLHL20 in ubiquitination.

## Contribution

The study identifies ZFTA as a novel KLHL20 substrate and provides insights into the fuzzy binding mechanism of disordered peptides.

## Key findings

- ZFTA peptides interact weakly and complexly with KLHL20Kelch, showing higher specificity than KLHL12Kelch.
- Peptide affinity is influenced by terminal residues, with N-terminal removal preserving affinity.
- The interaction mechanism is consistent with 'fuzzy binding', relevant to KLHL20's role in ubiquitination.

## Abstract

Interactions between peptides based on a region in the zinc finger translocation associated (ZFTA) protein and the Kelch domain of Kelch‐like protein 20 (KLHL20Kelch) have been characterised by biosensor analysis, supported by AlphaFold2‐based structure predictions of peptides bound to the protein. Residues critical for the interaction were identified. The analysis showed that all peptides exhibited relatively weak and complex interactions with KLHL20Kelch. The original ZFTA peptide had a much higher affinity for KLHL20Kelch than for the Kelch domain of KLHL12 (KLHL12Kelch), indicating a specificity for KLHL20Kelch. The estimated K
D
app of 35 µM was like that for a 21‐mer peptide derived from death‐associated protein kinase 1, a known KLHL20 substrate. Removal of flexible C‐terminal residues generated a 12‐mer, predicted to form a stable helix. This reduced the affinity 100‐fold. Removal of N‐terminal residues resulted in a 10‐mer predicted to be flexible, which had a similar affinity as the original 16‐mer. The similar affinities for peptides representing different regions of ZFTA suggest that the recognition is feature specific rather than sequence specific. The interaction mechanism reflects “fuzzy binding”, consistent with the role of KLHL20 as an adaptor protein in the ubiquitination of disordered protein substrates by Cullin‐3 E3 ubiquitin ligase.

An AlphaFold 2 model of a peptide derived from zinc finger translocation associated (ZFTA) protein in complex with the Kelch domain of KLHL20 (KLHL20Kelch). Sensorgrams from surface plasmon resonance biosensor experiments of the interaction between a ZFTA‐derived peptide with immobilised KLHL20Kelch shows that the interaction is complex and has a millimolar affinity. A fuzzy binding mechanism describes the complexities and specificity of the interaction.© 2026 WILEY‐VCH GmbH