Expanding the repertoire of usable E3 ubiquitin ligases remains a critical challenge in the field of targeted protein degradation. For the emerging E3 ligase Kelch domain-containing protein 2 (KLHDC2), currently reported ligands have a carboxylate moiety in common that mimics the natural degron but compromises cellular permeability. Here we report the discovery of carboxylate-free ligands for KLHDC2 through high-throughput screening and cellular functional evaluation. A fluorescence polarization-based screening assay identified NL1 as a micromolar KLHDC2 binder, which was confirmed by orthogonal biophysical methods. Although the structure-activity study did not improve affinity, a degron reporter assay demonstrated measurable cellular target engagement. Encouraged by these results, an expanded screen further identified NL2 as a neutral ligand with improved cellular activity. Computational analysis suggests that NL2 adopts a distinct binding mode, in which the conformational flexibility of two loops of KLHDC2 enables accommodation of its aromatic scaffold, while hydrophobic and π-driven interactions compensate for the canonical ionic contacts of other carboxylate-containing ligands. This work demonstrates that KLHDC2 can accommodate non-carboxylate scaffolds and expands the chemical space for developing KLHDC2-based targeted protein degradation strategies.
Yao Chen, Rui-Chen Gao, Jia-Jia Li et al.· Protein Science· 0 citations
This study addresses a major bottleneck in dairy waste management by engineering a highly efficient, thermophilic enzyme for lactose hydrolysis, transforming the cheese manufacturing by-product whey into a high-value resource. While traditional glycoside hydrolase family 2 (GH2) enzymes suffer from poor industrial thermostability, thermophilic GH42 alternatives lack the catalytic efficiency required for practical processing. To bridge this gap, a synergistic engineering strategy combining consensus sequence design and virtual binding energy screening was applied to Tn1577, a home-discovered GH42-galactosidase from Thermotoga naphthophila RUK10. Through homologous sequence analysis and iterative saturation mutagenesis, a superior triple mutant designated M10 (H271Q/V357G/Q340E) was successfully developed. Molecular dynamics (MD) simulations revealed that M10s performance is driven by 3 key structural alterations. First, a remodeled substrate tunnel accelerates lactose entry and product release while minimizing molecular congestion in the active cavity. Second, consensus mutations rigidify the catalytic pocket to preserve its geometry under thermal stress. Finally, a distal Q340E mutation rearranges local hydrogen bonding and forms a new anion-π interaction, anchoring the protein backbone to stabilize the overall conformation via allosteric regulation. This engineered rigidity introduces a beneficial kinetic trade-off: it drastically accelerates catalytic turnover by intentionally weakening substrate affinity, preventing enzyme stagnation and ensuring continuous catalytic cycling. Consequently, mutant M10 achieves a remarkable 6.5-fold increase in lactose hydrolysis activity at 55°C compared with the wild-type enzyme. Response surface methodology established ideal industrial parameters: 52.7°C, pH 5.5, and an enzyme dosage of 2.9 U/mL. Under these conditions, M10 accomplishes near-complete lactose hydrolysis of raw whey within a brief 2-h window. Operating at a temperature below the thermal unfolding point of core whey proteins (∼60°C), this mild thermal regimen shortens processing times and prevents the thermal denaturation of other valuable whey components, demonstrating M10s immense potential for high-efficiency, stable lactose-free dairy processing.
Si-Meng Ding, Jia-Jia Li, Jia-Hui Li et al.· Journal of Dairy Science· 0 citations
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