Multivalency achieves strong, yet reversible binding by the simultaneous formation of multiple weak bonds. It is a key interaction principle in biology and promising for the synthesis of ...high-affinity inhibitors of pathogens. We present a molecular model for the binding affinity of synthetic multivalent ligands onto multivalent receptors consisting of n receptor units arranged on a regular polygon. Ligands consist of a geometrically matching rigid polygonal core to which monovalent ligand units are attached via flexible linker polymers, closely mimicking existing experimental designs. The calculated binding affinities quantitatively agree with experimental studies for cholera toxin (n = 5) and anthrax receptor (n = 7) and allow to predict optimal core size and optimal linker length. Maximal binding affinity is achieved for a core that matches the receptor size and for linkers that have an equilibrium end-to-end distance that is slightly longer than the geometric separation between ligand core and receptor sites. Linkers that are longer than optimal are greatly preferable compared to shorter linkers. The angular steric restriction between ligand unit and linker polymer is shown to be a key parameter. We construct an enhancement diagram that quantifies the multivalent binding affinity compared to monovalent ligands. We conclude that multivalent ligands against influenza viral hemagglutinin (n = 3), cholera toxin (n = 5), and anthrax receptor (n = 7) can outperform monovalent ligands only for a monovalent ligand affinity that exceeds a core-size dependent threshold value. Thus, multivalent drug design needs to balance core size, linker length, as well as monovalent ligand unit affinity.
Multivalency is a key principle in reinforcing reversible molecular interactions through the formation of multiple bonds. The influenza A virus deploys this strategy to bind strongly to cell surface ...receptors. We performed single-molecule force spectroscopy (SMFS) to investigate the rupture force required to break individual and multiple bonds formed between synthetic sialic acid (SA) receptors and the two principal spike proteins of the influenza A virus (H3N2): hemagglutinin (H3) and neuraminidase (N2). Kinetic parameters such as the rupture length (χβ) and dissociation rate (k off) are extracted using the model by Friddle, De Yoreo, and Noy. We found that a monovalent SA receptor binds to N2 with a significantly higher bond lifetime (270 ms) compared to that for H3 (36 ms). By extending the single-bond rupture analysis to a multibond system of n protein-receptor pairs, we provide an unprecedented quantification of the mechanistic features of multivalency between H3 and N2 with SA receptors and show that the stability of the multivalent connection increases with the number of bonds from tens to hundreds of milliseconds. Association rates (k on) are also provided, and an estimation of the dissociation constants (K D) between the SA receptors to both proteins indicate a 17-fold higher binding affinity for the SA–N2 bond with respect to that of SA–H3. An optimal designed multivalent SA receptor showed a higher binding stability to the H3 protein of the influenza A virus than to the monovalent SA receptor. Our study emphasizes the influence of the scaffold on the presentation of receptors during multivalent binding.
Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are ...lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size-dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng–Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses.
As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane ...to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III–depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.
Alphaviruses
are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the
Alphaviru
s chikungunya virus (CHIKV) has spread explosively in the ...last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.
Cellular compartments are membrane-enclosed, spatially distinct microenvironments that confine and protect biochemical reactions in the biological cell. On the early Earth, the autonomous formation ...of compartments is thought to have led to the encapsulation of nucleotides, thereby satisfying a starting condition for the emergence of life. Recently, surfaces have come into focus as potential platforms for the self-assembly of prebiotic compartments, as significantly enhanced vesicle formation was reported in the presence of solid interfaces. The detailed mechanism of such formation at the mesoscale is still under discussion. We report here on the spontaneous transformation of solid-surface-adhered lipid deposits to unilamellar membrane compartments through a straightforward sequence of topological changes, proceeding via a network of interconnected lipid nanotubes. We show that this transformation is entirely driven by surface-free energy minimization and does not require hydrolysis of organic molecules or external stimuli such as electrical currents or mechanical agitation. The vesicular structures take up and encapsulate their external environment during formation and can subsequently separate and migrate upon exposure to hydrodynamic flow. This may link the self-directed transition from weakly organized bioamphiphile assemblies on solid surfaces to protocells with secluded internal contents.
We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general ...rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.
The force-induced desorption of single peptide chains from mixed OH/CH3-terminated self-assembled monolayers is studied in closely matched molecular dynamics simulations and atomic force microscopy ...experiments with the goal to gain microscopic understanding of the transition between peptide adsorption and adsorption resistance as the surface contact angle is varied. In both simulations and experiments, the surfaces become adsorption resistant against hydrophilic as well as hydrophobic peptides when their contact angle decreases below θ ≈ 50°–60°, thus confirming the so-called Berg limit established in the context of protein and cell adsorption. Entropy/enthalpy decomposition of the simulation results reveals that the key discriminator between the adsorption of different residues on a hydrophobic monolayer is of entropic nature and thus is suggested to be linked to the hydrophobic effect. By pushing a polyalanine peptide onto a polar surface, simulations reveal that the peptide adsorption resistance is caused by the strongly bound water hydration layer and characterized by the simultaneous gain of both total entropy in the system and total number of hydrogen bonds between water, peptide, and surface. This mechanistic insight into peptide adsorption resistance might help to refine design principles for anti-fouling surfaces.
Objectives Investigations of cone beam computed tomography (CBCT) for bisphosphonate-related osteonecrosis of the jaw (BRONJ) imaging are rare. The purpose of this study was to investigate the ...prevalence of typical radiological findings of BRONJ in CBCT. Methods Twenty-seven CBCTs of BRONJ sites were assessed on the basis of the radiological findings (cancellous bone destruction, cortical bone erosion, sequestration, osteosclerosis, and periostal bone formation) and put in relation to the severity of the BRONJ sites. Results Cancellous bone destruction and cortical bone erosion were the most common findings. Occurrence seems to decrease with decreasing BRONJ severity. Sequestration and osteosclerosis were less frequent and could be seen across all stages. Periosteal bone formation occurred in high-stage BRONJ only. Conclusion Cancellous bone destruction, cortical bone erosion, sequestration, and osteosclerosis can be seen across all stages and prevalence seems to decrease with decreasing severity of BRONJ. The occurrence of periosteal new bone formation seems to start in high-stage BRONJ.
Herein, the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing oligoethylene glycol (OEG) spacers interacting with spike proteins of ...influenza A virus (IAV) X31 is described. Although the flexible Tris‐based trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well as biophysical analysis, these findings emphasize on the importance of the scaffold rigidity and the challenges associated with the spacer length optimization.
The rigid adamantane‐based trivalent sialoside with optimized oligoethylene glycol spacer was found to be the most potent candidate against IAV/X31, showing an inhibitory constant in the micromolar range. It binds to hemagglutinin of the influenza virus and thus inhibits the virus–cell attachment.
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