Biology subjects

Üffing, A.

Publications and source records attributed to Üffing, A..

4 recordsLinked to original sources

bioRxiv2026

BCAS3 regulates WIPI2b via a direct interaction in the ATG16L1-binding cleft

De novo formation of the phagophore membrane is a fundamental step in autophagy and relies on recruitment of the E3-like ligase complex via an interaction between ATG16L1 and phosphatidylinositol 3-phosphate (PtdIns3P)-effector protein, WIPI2b. Although WIPI2b can directly bind membranes, how its membrane association is regulated is still not fully understood. Here, we describe BCAS3 as a novel direct interactor of WIPI2b and demonstrate that a peptide of BCAS3 binds in the ATG16L1-binding cleft of WIPI2b with low micromolar affinity. We show that BCAS3 and its partner PHAF1 induce recruitment of WIPI2b to membranes via the interaction of BCAS3 with PtdIns3P. This not only challenges the current paradigm, whereby ATG16L1 is the only confirmed binder of WIPI2b in this region, but also offers a mechanism through which WIPI2b membrane association might be regulated.

cell biology↗

bioRxiv2024

Accurate de novo design of high-affinity protein binding macrocycles using deep learning

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate KD between 1-10 M, and the best anti-GABARAP macrocycle binds with a KD of 6 nM and a sub-nanomolar IC50 in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with KD < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 [A] to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

biochemistry↗

bioRxiv2024

EGFR meets hATG8s - Biophysical and structural insights supporting a unique role of GABARAP during receptor trafficking

The human ATG8 family member GABARAP is involved in numerous autophagy-related and - unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances EGFR degradation upon ligand stimulation. Here, we report on two putative LC3-interacting regions (LIRs) within the EGFR, the first of which (LIR1) is selected as GABARAP binding site in-silico. Indeed, in-vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X-ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting a canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding.

biophysics↗

bioRxiv2024

Exploring Arylidene-Indolinone Ligands of Autophagy Proteins LC3B and GABARAP

We report the first structure-activity studies of arylidene-indolinone compound GW5074 which was reported as a ligand of autophagy-related protein LC3B. The literature has conflicting information on the binding affinity of this compound and there is some debate regarding its use as a component of autophagy-dependent degrader compounds. We developed an AlphaScreen assay to measure competitive inhibition of the binding of known peptide ligands to LC3B and its paralog GABARAP. 18 analogs were synthesized and tested against both proteins. Inhibitory potencies were found to be in the mid- to high micromolar range. 2D-NMR data revealed the binding site on GABARAP as hydrophobic pocket 1, where native peptide ligands bind with an aromatic side chain. Our results suggest that GW5074 binds LC3B and GABARAP with micromolar affinity. These affinities could support further exploration in targeted protein degradation, but only if off-target effects and poor solubility can be appropriately addressed.

biochemistry↗