Advanced applications of Nanodiscs-based platforms for antibodies discovery.

Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs.

Structural insights into thrombolytic activity of destabilase from medicinal leech.

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

All-d-Enantiomeric Peptide D3 Designed for Alzheimer's Disease Treatment Dynamically Interacts with Membrane-Bound Amyloid-ß Precursors.

Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-ß peptide (Aß) oligomers play a crucial role in AD pathogenesis. All-d-Enantiomeric peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aß aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aß monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aß sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aß conversion into ß-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.

NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs.

P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism.