Hepatitis C virus (HCV) affects 47 million people and causes 239,000 deaths annually, yet no vaccine is on the horizon. Generating a stable native-like mimic of the envelope complex E1E2, the only target for known neutralizing antibodies, is an important aim for HCV vaccine development. Starting from a recombinant E1E2 design that utilizes a leucine zipper for proper folding, we used an iterative structure-based design approach to engineer antigens with at least 100-fold stronger binding to conformational antibodies and increased thermal stability. These new E1E2 designs facilitate production of native-like E1E2 antigens based on strains from different HCV genotypes and enable the generation of a recombinant E1E2 antigen design that lacks the immunogenic leucine zipper. A cryo-EM structure of one of the stabilized E1E2 antigens in complex with neutralizing antibody AT1211 provides atomic-level insights into an atypical epitope on the E2 subunit. Finally, immunogenicity studies in rabbits with adjuvanted E1E2 proteins show that immunogen stabilization alone does do not enhance serum neutralization breadth, but that removing the leucine zipper does increase homologous serum neutralization. The hepatitis C virus E1E2 glycoprotein is the only target of neutralizing antibodies. Here, the authors used structure-based design to generate soluble antigens that resemble the viral E1E2 heterodimer.
Joan Capella-Pujol, Fabian Mulder, F. Cannac et al.· Nature Communications· 1 citation
PIEZO channels are mechanosensitive membrane proteins whose activation is governed by the surrounding lipid environment. However, the direct mechanistic contribution of native membrane composition to the molecular interactions remains unclear. In this study, a systematic comparison is made between PIEZO1 reconstituted in detergent micelles and in cell membrane–derived nanodiscs, which preserve the native lipid composition. Initial characterization employing a combination of atomic force microscopy and coarse-grained molecular dynamics simulations unveils distinct physical signatures of PIEZO1 in these two environments. Single-molecule force spectroscopy measurements demonstrate that interaction between the extracellular domain of PIEZO1 and a specific antibody exhibits unique mechanical responses strongly influenced by the surrounding membrane. In nanodiscs, PIEZO1 exhibits reversible, elastic-like behavior with preserved structural integrity and consistent adhesion forces even when modulated by Yoda1 (agonist) and Dooku1 (antagonist). Conversely, micelles induce a plastic response with altered mechanosensitivity and functional stability. Based on these findings, we propose a possible membrane-mediated force transmission pathway and quantify a simplified interaction energy landscape. Collectively, our findings offer the initial direct evidence of how the native lipid environment mechanistically governs PIEZO1 interactions, establishing native membranes as critical determinants for mechanotransduction.
Sanjai Karanth, Alessandro Nicoli, F. Cannac et al.· bioRxiv· 0 citations
