Biology subjects

Wallden, K.

Publications and source records attributed to Wallden, K..

3 recordsLinked to original sources

bioRxiv2025

Structure of the central Staphylococcus aureus AAA+ protease MecA/ClpC/ClpP

Bacterial AAA+ proteases are composed of a AAA+ partner (e.g. ClpC) and an associated peptidase (e.g. ClpP). They represent ATP-fuelled and self-compartmentalized proteolytic machines that are crucial for stress resistance and virulence. ClpC requires cooperation with adaptor proteins such as MecA for activation and complex formation with ClpP. Here, we present the cryo-EM structure of the MecA/ClpC/ClpP complex from the major pathogen Staphylococcus aureus. MecA forms a dynamic crown on top of the ClpC/ClpP complex with its substrate-binding domain positioned near the ClpC pore site, likely facilitating substrate transfer. ClpC/ClpP complex formation involves ClpC P-loops and ClpP N-terminal {beta}-hairpins, which insert into the central ClpC threading channel and contact sites next to the ClpC ATPase center. ClpC and ClpP interactions are asymmetric and dictated by the activity states of ClpC ATPase subunits. ClpP binding increases ClpC ATPase and threading activities in a {beta}-hairpin dependent manner, illuminating an allosteric pathway in the cooperation of ATPase and peptidase components in bacterial AAA+ proteases.

biochemistry↗

bioRxiv2025

Development of DARPin T cell engagers for specific targeting of tumor-associated HLA/peptide complexes

The compromise between affinity and specificity in TCR-dependent targeting of HLA-restricted tumor-associated antigens presents a significant challenge in developing efficacious immunotherapies. As such, T cell engagers which circumvent these limitations are of particular interest. We have established a process to generate bispecific Designed Ankyrin Repeat Proteins (DARPins) that simultaneously target HLA-I molecules in complex with tumor-associated peptides and CD3{varepsilon}. High-affinity HLA-A*0201/NY-ESO1157-165-specific DARPins were isolated after only four rounds of in-vitro selection from naive DARPin libraries. Combining HLA-A*0201/NY-ESO1157-165-specific DARPins with a CD3{varepsilon}-specific DARPin created potent T cell engagers which elicited CD8+ T cell activation towards tumor targets with high peptide specificity, as confirmed by X-scanning mutagenesis and functional killing assays. The cryo-EM structure of a ternary DARPin/HLA-A*0201/NY-ESO1157-165 complex revealed a rigid and concave DARPin surface that binds to the entire length of the peptide-binding cleft, contacting both -helices and the peptide. The present results unveil promising immuno-oncotherapeutic approaches with the possibility of rapidly developing DARPins with high affinity and specificity to HLA/peptide targets that can be readily combined with a new generation of anti-CD3{varepsilon}-specific DARPins.

synthetic biology↗

bioRxiv2019

A simple pressure-assisted method for cryo-EM specimen preparation

Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal contents. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.

molecular biology↗