Deconstructing SARS-CoV-2 neutralization: A modular molecular framework for computational design and comparison of antibodies and nanobodies targeting the spike RBD.

Since 2020 the COVID-19 pandemic has led scientists to search for strategies to predict the transmissibility and virulence of new severe acute respiratory syndrome coronavirus 2 variants based on the estimation of the affinity of the spike receptor binding domain (RBD) for the human angiotensin-converting enzyme 2 (ACE2) receptor and/or neutralizing antibodies. In this context, our lab developed a computational pipeline to quickly quantify the free energy of interaction at the spike RBD/ACE2 protein-protein interface, reflecting the incidence trend observed in the transmissibility/virulence of the investigated variants. In this new study, we used our pipeline to estimate the free energy of interaction between the RBD from 10 variants, and 14 antibodies (ab), or 5 nanobodies (nb), highlighting the RBD regions preferentially targeted by the investigated ab/nb. Our structural comparative analysis and interaction energy calculations allowed us to propose the most promising RBD regions to be targeted by future ab/nb to be designed by site-directed mutagenesis of existing high-affinity ab/nb, to increase their affinity for the target RBD region, for preventing spike-RBD/ACE2 interactions and virus entry in host cells. Furthermore, we evaluated the ability of the investigated ab/nb to simultaneously interact with the three RBD located on the surface of the trimeric spike protein, which can alternatively be in up- or down- (all-3-up-, all-3-down-, 1-up-/2-down-, 2-up-/1-down-) conformations.

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells.

Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of kon (linear to bent) from 3.2 to 10.7 s-1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

Antibody-induced crosslinking and cholesterol-sensitive, anomalous diffusion of nicotinic acetylcholine receptors.

Synaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs, for example in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of monoclonal antibody-crosslinked receptors in the clonal cell line CHO-K1/A5, which robustly expresses adult muscle-type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (~34 nm radius, ~400 ms lifetime). Dynamic clustering, trapping, and immobilization also occur in larger nanocluster zones (120-180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of monoclonal antibody-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia.

Conformational plasticity at the IgE-binding site of the B-cell receptor CD23.

IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.