Protein dynamics provide mechanistic insights about epistasis among common missense polymorphisms.

Sequencing of the protein coding genome has revealed many different missense mutations of human proteins and different population frequencies of corresponding haplotypes, which consist of different sets of those mutations. Here, we present evidence for pairwise intramolecular epistasis (i.e., nonadditive interactions) between many such mutations through an analysis of protein dynamics. We suggest that functional compensation for conserving protein dynamics is a likely evolutionary mechanism that maintains high-frequency mutations that are individually nonneutral but epistatically compensating within proteins. This analysis is the first of its type to look at human proteins with specific high population frequency mutations and examine the relationship between mutations that make up that observed high-frequency protein haplotype. Importantly, protein dynamics revealed a separation between high and low frequency haplotypes within a target protein cytochrome P450 2A7, with the high-frequency haplotypes showing behavior closer to the wild-type protein. Common protein haplotypes containing two mutations display dynamic compensation in which one mutation can correct for the dynamic effects of the other. We also utilize a dynamics-based metric, EpiScore, that evaluates the epistatic interactions and allows us to see dynamic compensation within many other proteins.

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants.

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of ß-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.

Dynamic allostery highlights the evolutionary differences between the CoV-1 and CoV-2 main proteases.

The SARS-CoV-2 coronavirus has become one of the most immediate and widely studied systems since its identification and subsequent global outbreak from 2019 to 2022. In an effort to understand the biophysical changes as a result of mutations, the mechanistic details of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we explored the long-range dynamics using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter- and intrachain, for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and specific distal residues in the CoV-2 mPro not present in CoV-1. The enhanced long-distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the dynamic flexibility index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1, while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, in which the new substitutions in CoV-2 induce flexibility and dynamic changes elsewhere in the structure.

Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions.

Amino acid substitutions at nonconserved protein positions can have noncanonical and "long-distance" outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions ("toggle"), ten nonconserved positions showed progressive changes from a range of substitutions ("rheostat"), and three nonconserved positions tolerated almost all substitutions ("neutral"). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

The Role of Rigid Residues in Modulating TEM-1 ß-Lactamase Function and Thermostability.

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in ß-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A ß-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on ß-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of ß-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

Allostery and Epistasis: Emergent Properties of Anisotropic Networks.

Understanding the underlying mechanisms behind protein allostery and non-additivity of substitution outcomes (i.e., epistasis) is critical when attempting to predict the functional impact of mutations, particularly at non-conserved sites. In an effort to model these two biological properties, we extend the framework of our metric to calculate dynamic coupling between residues, the Dynamic Coupling Index (DCI) to two new metrics: (i) EpiScore, which quantifies the difference between the residue fluctuation response of a functional site when two other positions are perturbed with random Brownian kicks simultaneously versus individually to capture the degree of cooperativity of these two other positions in modulating the dynamics of the functional site and (ii) DCIasym, which measures the degree of asymmetry between the residue fluctuation response of two sites when one or the other is perturbed with a random force. Applied to four independent systems, we successfully show that EpiScore and DCIasym can capture important biophysical properties in dual mutant substitution outcomes. We propose that allosteric regulation and the mechanisms underlying non-additive amino acid substitution outcomes (i.e., epistasis) can be understood as emergent properties of an anisotropic network of interactions where the inclusion of the full network of interactions is critical for accurate modeling. Consequently, mutations which drive towards a new function may require a fine balance between functional site asymmetry and strength of dynamic coupling with the functional sites. These two tools will provide mechanistic insight into both understanding and predicting the outcome of dual mutations.

Coevolving residues inform protein dynamics profiles and disease susceptibility of nSNVs.

The conformational dynamics of proteins is rarely used in methodologies used to predict the impact of genetic mutations due to the paucity of three-dimensional protein structures as compared to the vast number of available sequences. Until now a three-dimensional (3D) structure has been required to predict the conformational dynamics of a protein. We introduce an approach that estimates the conformational dynamics of a protein, without relying on structural information. This de novo approach utilizes coevolving residues identified from a multiple sequence alignment (MSA) using Potts models. These coevolving residues are used as contacts in a Gaussian network model (GNM) to obtain protein dynamics. B-factors calculated using sequence-based GNM (Seq-GNM) are in agreement with crystallographic B-factors as well as theoretical B-factors from the original GNM that utilizes the 3D structure. Moreover, we demonstrate the ability of the calculated B-factors from the Seq-GNM approach to discriminate genomic variants according to their phenotypes for a wide range of proteins. These results suggest that protein dynamics can be approximated based on sequence information alone, making it possible to assess the phenotypes of nSNVs in cases where a 3D structure is unknown. We hope this work will promote the use of dynamics information in genetic disease prediction at scale by circumventing the need for 3D structures.

Mutations Utilize Dynamic Allostery to Confer Resistance in TEM-1 ß-lactamase.

ß-lactamases are enzymes produced by bacteria to hydrolyze ß-lactam antibiotics as a common mechanism of resistance. Evolution in such enzymes has been rendering a wide variety of antibiotics impotent, therefore posing a major threat. Clinical and in vitro studies of evolution in TEM-1 ß-lactamase have revealed a large number of single point mutations that are responsible for driving resistance to antibiotics and/or inhibitors. The distal locations of these mutations from the active sites suggest that these allosterically modulate the antibiotic resistance. We investigated the effects of resistance driver mutations on the conformational dynamics of the enzyme to provide insights about the mechanism of their long-distance interactions. Through all-atom molecular dynamics (MD) simulations, we obtained the dynamic flexibility profiles of the variants and compared those with that of the wild type TEM-1. While the mutational sites in the variants did not have any direct van der Waals interactions with the active site position S70 and E166, we observed a change in the flexibility of these sites, which play a very critical role in hydrolysis. Such long distance dynamic interactions were further confirmed by dynamic coupling index (DCI) analysis as the sites involved in resistance driving mutations exhibited high dynamic coupling with the active sites. A more exhaustive dynamic analysis, using a selection pressure for ampicillin and cefotaxime resistance on all possible types of substitutions in the amino acid sequence of TEM-1, further demonstrated the observed mechanism. Mutational positions that play a crucial role for the emergence of resistance to new antibiotics exhibited high dynamic coupling with the active site irrespective of their locations. These dynamically coupled positions were neither particularly rigid nor particularly flexible, making them more evolvable positions. Nature utilizes these sites to modulate the dynamics of the catalytic sites instead of mutating the highly rigid positions around the catalytic site.

Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme.

ß-Lactamases are produced by many modern bacteria as a mechanism of resistance toward ß-lactam antibiotics, the most common antibiotics in use. ß-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A ß-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern ß-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant ß-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient ß-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

Adaptive BP-Dock: An Induced Fit Docking Approach for Full Receptor Flexibility.

We present an induced fit docking approach called Adaptive BP-Dock that integrates perturbation response scanning (PRS) with the flexible docking protocol of RosettaLigand in an adaptive manner. We first perturb the binding pocket residues of a receptor and obtain a new conformation based on the residue response fluctuation profile using PRS. Next, we dock a ligand to this new conformation by RosettaLigand, where we repeat these steps for several iterations. We test this approach on several protein test sets including difficult unbound docking cases such as HIV-1 reverse transcriptase and HIV-1 protease. Adaptive BP-Dock results show better correlation with experimental binding affinities compared to other docking protocols. Overall, the results imply that Adaptive BP-Dock can easily capture binding induced conformational changes by simultaneous sampling of protein and ligand conformations. This can provide faster and efficient docking of novel targets for rational drug design.

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin.

Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), ß-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.

A Rigid Hinge Region Is Necessary for High-Affinity Binding of Dimannose to Cyanovirin and Associated Constructs.

Mutations in the hinge region of cyanovirin-N (CVN) dictate its preferential oligomerization state. Constructs with the Pro51Gly mutation preferentially exist as monomers, whereas wild-type cyanovirin can form domain-swapped dimers under certain conditions. Because the hinge region is an integral part of the high-affinity binding site of CVN, we investigated whether this mutation affects the shape, flexibility, and binding affinity of domain B for dimannose. Our studies indicate that the capability of monomeric wild-type CVN to resist mechanical perturbations is enhanced when compared to that of constructs in which the hinge region is more flexible. Our computational results also show that enhanced flexibility leads to blocking of the binding site by allowing different rotational isomeric states of Asn53. Moreover, at higher temperatures, this observed flexibility leads to an interaction between Asn53 and Asn42, further hindering access to the binding site. On the basis of these results, we predicted that binding affinity for dimannose would be more favorable for cyanovirin constructs containing a wild-type hinge region, whereas affinity would be impaired in the case of mutants containing Pro51Gly. Experimental characterization by isothermal titration calorimetry of a set of cyanovirin mutants confirms this hypothesis. Those possessing the Pro51Gly mutation are consistently inferior binders.

Partial unfolding and refolding for structure refinement: A unified approach of geometric simulations and molecular dynamics.

The most successful protein structure prediction methods to date have been template-based modeling (TBM) or homology modeling, which predicts protein structure based on experimental structures. These high accuracy predictions sometimes retain structural errors due to incorrect templates or a lack of accurate templates in the case of low sequence similarity, making these structures inadequate in drug-design studies or molecular dynamics simulations. We have developed a new physics based approach to the protein refinement problem by mimicking the mechanism of chaperons that rehabilitate misfolded proteins. The template structure is unfolded by selectively (targeted) pulling on different portions of the protein using the geometric based technique FRODA, and then refolded using hierarchically restrained replica exchange molecular dynamics simulations (hr-REMD). FRODA unfolding is used to create a diverse set of topologies for surveying near native-like structures from a template and to provide a set of persistent contacts to be employed during re-folding. We have tested our approach on 13 previous CASP targets and observed that this method of folding an ensemble of partially unfolded structures, through the hierarchical addition of contact restraints (that is, first local and then nonlocal interactions), leads to a refolding of the structure along with refinement in most cases (12/13). Although this approach yields refined models through advancement in sampling, the task of blind selection of the best refined models still needs to be solved. Overall, the method can be useful for improved sampling for low resolution models where certain of the portions of the structure are incorrectly modeled.

Conformational dynamics of nonsynonymous variants at protein interfaces reveals disease association.

Recent studies have shown that the protein interface sites between individual monomeric units in biological assemblies are enriched in disease-associated non-synonymous single nucleotide variants (nsSNVs). To elucidate the mechanistic underpinning of this observation, we investigated the conformational dynamic properties of protein interface sites through a site-specific structural dynamic flexibility metric (dfi) for 333 multimeric protein assemblies. dfi measures the dynamic resilience of a single residue to perturbations that occurred in the rest of the protein structure and identifies sites contributing the most to functionally critical dynamics. Analysis of dfi profiles of over a thousand positions harboring variation revealed that amino acid residues at interfaces have lower average dfi (31%) than those present at non-interfaces (50%), which means that protein interfaces have less dynamic flexibility. Interestingly, interface sites with disease-associated nsSNVs have significantly lower average dfi (23%) as compared to those of neutral nsSNVs (42%), which directly relates structural dynamics to functional importance. We found that less conserved interface positions show much lower dfi for disease nsSNVs as compared to neutral nsSNVs. In this case, dfi is better as compared to the accessible surface area metric, which is based on the static protein structure. Overall, our proteome-wide conformational dynamic analysis indicates that certain interface sites play a critical role in functionally related dynamics (i.e., those with low dfi values), therefore mutations at those sites are more likely to be associated with disease.

A flexible docking scheme efficiently captures the energetics of glycan-cyanovirin binding.

Cyanovirin-N (CVN), a cyanobacterial lectin, exemplifies a class of antiviral agents that inhibit HIV by binding to the highly glycosylated envelope protein gp120. Here, we investigate the energetics of glycan recognition using a computationally inexpensive flexible docking approach, backbone perturbation docking (BP-Dock). We benchmarked our method using two mutants of CVN: P51G-m4-CVN, which binds dimannose with high affinity through domain B, and CVN((mutDB)), in which binding to domain B has been abolished through mutation of five polar residues to small nonpolar side chains. We investigated the energetic contribution of these polar residues along with the additional position 53 by docking dimannose to single-point CVN mutant models. Analysis of the docking simulations indicated that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen-bond network that reverberated throughout the binding cavity. N42A decreased the binding score to a level comparable to that of CVN((mutDB)) by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score, similar to P51G-m4-CVN. Experimental characterization of the five mutants by NMR spectroscopy confirmed the binding affinity pattern predicted by BP-Dock. Despite their mostly conserved fold and stability, E41A, E41G, and T57A displayed dissociation constants in the millimolar range. N53S showed a binding constant in the low micromolar range, similar to that observed for P51G-m4-CVN. No binding was observed for N42A. Our results show that BP-Dock is a useful tool for rapidly screening the relative binding affinity pattern of in silico-designed mutants compared with wild-type, supporting its use to design novel mutants with enhanced binding properties.

Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation.

Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP-ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP-ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP-ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance.

BP-Dock: a flexible docking scheme for exploring protein-ligand interactions based on unbound structures.

Molecular docking serves as an important tool in modeling protein-ligand interactions. However, it is still challenging to incorporate overall receptor flexibility, especially backbone flexibility, in docking due to the large conformational space that needs to be sampled. To overcome this problem, we developed a novel flexible docking approach, BP-Dock (Backbone Perturbation-Dock) that can integrate both backbone and side chain conformational changes induced by ligand binding through a multi-scale approach. In the BP-Dock method, we mimic the nature of binding-induced events as a first-order approximation by perturbing the residues along the protein chain with a small Brownian kick one at a time. The response fluctuation profile of the chain upon these perturbations is computed using the perturbation response scanning method. These response fluctuation profiles are then used to generate binding-induced multiple receptor conformations for ensemble docking. To evaluate the performance of BP-Dock, we applied our approach on a large and diverse data set using unbound structures as receptors. We also compared the BP-Dock results with bound and unbound docking, where overall receptor flexibility was not taken into account. Our results highlight the importance of modeling backbone flexibility in docking for recapitulating the experimental binding affinities, especially when an unbound structure is used. With BP-Dock, we can generate a wide range of binding site conformations realized in nature even in the absence of a ligand that can help us to improve the accuracy of unbound docking. We expect that our fast and efficient flexible docking approach may further aid in our understanding of protein-ligand interactions as well as virtual screening of novel targets for rational drug design.

Evidence that the cold- and menthol-sensing functions of the human TRPM8 channel evolved separately.

Transient receptor potential melastatin 8 (TRPM8) is a temperature- and menthol-sensitive ion channel that contributes to diverse physiological roles, including cold sensing and pain perception. Clinical trials targeting TRPM8 have faced repeated setbacks predominantly due to the knowledge gap in unraveling the molecular underpinnings governing polymodal activation. A better understanding of the molecular foundations between the TRPM8 activation modes may aid the development of mode-specific, thermal-neutral therapies. Ancestral sequence reconstruction was used to explore the origins of TRPM8 activation modes. By resurrecting key TRPM8 nodes along the human evolutionary trajectory, we gained valuable insights into the trafficking, stability, and function of these ancestral forms. Notably, this approach unveiled the differential emergence of cold and menthol sensitivity over evolutionary time, providing a fresh perspective on complex polymodal behavior. These studies provide a paradigm for understanding polymodal behavior in TRPM8 and other proteins with the potential to enhance our understanding of sensory receptor biology and pave the way for innovative therapeutic interventions.

The antiviral lectin cyanovirin-N: probing multivalency and glycan recognition through experimental and computational approaches.

CVN (cyanovirin-N), a small lectin isolated from cyanobacteria, exemplifies a novel class of anti-HIV agents that act by binding to the highly glycosylated envelope protein gp120 (glycoprotein 120), resulting in inhibition of the crucial viral entry step. In the present review, we summarize recent work in our laboratory and others towards determining the crucial role of multivalency in the antiviral activity, and we discuss features that contribute to the high specificity and affinity for the glycan ligand observed in CVN. An integrated approach that encompasses structural determination, mutagenesis analysis and computational work holds particular promise to clarify aspects of the interactions between CVN and glycans.