Many cancers overexpress one or more of the six human pro-survival BCL2 family proteins to evade apoptosis. To determine which BCL2 protein or proteins block apoptosis in different cancers, we computationally designed three-helix bundle protein inhibitors specific for each BCL2 pro-survival protein. Following in vitro optimization, each inhibitor binds its target with high picomolar to low nanomolar affinity and at least 300-fold specificity. Expression of the designed inhibitors in human cancer cell lines revealed unique dependencies on BCL2 proteins for survival which could not be inferred from other BCL2 profiling methods. Our results show that designed inhibitors can be generated for each member of a closely-knit protein family to probe the importance of specific protein-protein interactions in complex biological processes.

Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of Kv7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of Kv7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP2), and our molecular dynamics simulation suggests PIP2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in Kv7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of Kv7-associated EE.

Vaccine hesitancy and continuing emergence of SARS-CoV-2 variants of concern that may escape vaccine-induced immune responses highlight the urgent need for effective COVID-19 therapeutics. Monoclonal antibodies used in the clinic have varying efficacies against distinct SARS-CoV-2 variants; thus, there is considerable interest in engineered ACE2 peptides with augmented binding affinities for SARS-CoV-2 Spike protein. These could have therapeutic benefit against multiple viral variants. Using molecular dynamics simulations, we show how three amino acid substitutions in an engineered soluble ACE2 peptide (sACE2 2 .v2.4-IgG1) markedly increase affinity for the SARS-CoV-2 Spike (S) protein. We demonstrate high binding affinity to S protein of the early SARS-CoV-2 WA-1/2020 isolate and also to multiple variants of concern: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) SARS-CoV-2 variants. In humanized K18-hACE2 mice, prophylactic and therapeutic administration of sACE2 2 .v2.4-IgG1 peptide prevented acute lung vascular endothelial injury and lung edema (essential features of ARDS) and significantly improved survival after infection by SARS-CoV-2 WA-1/2020 as well as P.1 variant of concern. These studies demonstrate for the first time broad efficacy in vivo of an ACE2 decoy peptide against multiple SARS-CoV-2 variants and point to its therapeutic potential.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

Deep mutational scanning or deep mutagenesis is a powerful tool for understanding the sequence diversity available to viruses for adaptation in a laboratory setting. It generally involves tracking an in vitro selection of protein sequence variants with deep sequencing to map mutational effects based on changes in sequence abundance. Coupled with any of a number of selection strategies, deep mutagenesis can explore the mutational diversity available to viral glycoproteins, which mediate critical roles in cell entry and are exposed to the humoral arm of the host immune response. Mutational landscapes of viral glycoproteins for host cell attachment and membrane fusion reveal extensive epistasis and potential escape mutations to neutralizing antibodies or other therapeutics, as well as aiding in the design of optimized immunogens for eliciting broadly protective immunity. While less explored, deep mutational scans of host receptors further assist in understanding virus-host protein interactions. Critical residues on the host receptors for engaging with viral spikes are readily identified and may help with structural modeling. Furthermore, mutations may be found for engineering soluble decoy receptors as neutralizing agents that specifically bind viral targets with tight affinity and limited potential for viral escape. By untangling the complexities of how sequence contributes to viral glycoprotein and host receptor interactions, deep mutational scanning is impacting ideas and strategies at multiple levels for combatting circulating and emergent virus strains.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find an engineered decoy receptor, sACE2 2 .v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. Variant N501Y in the RBD, which has emerged in a rapidly spreading lineage (B.1.1.7) in England, enhances affinity for wild type ACE2 20-fold but remains tightly bound to engineered sACE22.v2.4. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

In response to touch, some carnivorous plants such as the Venus flytrap have evolved spectacular movements to capture animals for nutrient acquisition. However, the molecules that confer this sensitivity remain unknown. We used comparative transcriptomics to show that expression of three genes encoding homologs of the MscS-Like (MSL) and OSCA/TMEM63 family of mechanosensitive ion channels are localized to touch-sensitive trigger hairs of Venus flytrap. We focus here on the candidate with the most enriched expression in trigger hairs, the MSL homolog FLYCATCHER1 (FLYC1). We show that FLYC1 transcripts are localized to mechanosensory cells within the trigger hair, transfecting FLYC1 induces chloride-permeable stretch-activated currents in naïve cells, and transcripts coding for FLYC1 homologs are expressed in touch-sensing cells of Cape sundew, a related carnivorous plant of the Droseraceae family. Our data suggest that the mechanism of prey recognition in carnivorous Droseraceae evolved by co-opting ancestral mechanosensitive ion channels to sense touch.

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high-affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and Tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Based on structural and functional data, a loop sequence of variable lengths has been proposed to stabilize empty MHC-I molecules through direct interactions with the floor of the groove. Using deep mutagenesis on two complementary expression systems, we find that important residues for the Tapasin/TAPBPR chaperoning activity are located on a large scaffolding surface, excluding the loop. Conversely, loop mutations influence TAPBPR interactions with properly conformed MHC-I molecules, relevant for peptide editing. Detailed biophysical characterization by solution NMR, ITC and FP-based assays shows that the loop hovers above the MHC-I groove to promote the capture of incoming peptides. Our results suggest that the longer loop of TAPBPR lowers the affinity requirements for peptide selection to facilitate peptide loading under conditions and subcellular compartments of reduced ligand concentration, and to prevent disassembly of high-affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in leukocyte development and inflammation and their status as coreceptors for HIV-1 infection, among other roles. Both receptors form dimers or oligomers of unclear function. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate their dimerization interfaces, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that change how the receptors self-associate, either via specific oligomer assembly or alternative mechanisms of clustering in close proximity. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations predicted from the scan to reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. A mutation in the dimer interface of CXCR4 had increased binding to the ligand CXCL12 and yet diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens.

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states have provided a glimpse of the functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to intermediate states are not fully understood. Furthermore, the molecular basis of how serotonin is recognized and transported remains unclear. In this study, we performed unbiased microsecond-long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational-driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We find that the transition from the occluded to inward-facing state is the rate-limiting step for substrate import and that the substrate decreases the free energy barriers to achieve the inward-facing state. Our study provides insights on the molecular basis of dynamics-driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a 'decoy' to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy, FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments. FLIF displayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) against FLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys like FLIF may be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.

While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFRα carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application.

Even with the availability of vaccines, therapeutic options for COVID-19 still remain highly desirable, especially in hospitalized patients with moderate or severe disease. Soluble ACE2 (sACE2) is a promising therapeutic candidate that neutralizes SARS CoV-2 infection by acting as a decoy. Using computational mutagenesis, we designed a number of sACE2 derivatives carrying three to four mutations. The top-predicted sACE2 decoy based on the in silico mutagenesis scan was subjected to molecular dynamics and free-energy calculations for further validation. After illuminating the mechanism of increased binding for our designed sACE2 derivative, the design was verified experimentally by flow cytometry and BLI-binding experiments. The computationally designed sACE2 decoy (ACE2-FFWF) bound the receptor-binding domain of SARS-CoV-2 tightly with low nanomolar affinity and ninefold affinity enhancement over the wild type. Furthermore, cell surface expression was slightly greater than wild-type ACE2, suggesting that the design is well-folded and stable. Having an arsenal of high-affinity sACE2 derivatives will help to buffer against the emergence of SARS CoV-2 variants. Here, we show that computational methods have become sufficiently accurate for the design of therapeutics for current and future viral pandemics.

SARS coronavirus 2 is neutralized by proteins that block receptor-binding sites on spikes that project from the viral envelope. In particular, substantial research investment has advanced monoclonal antibody therapies to the clinic where they have shown partial efficacy in reducing viral burden and hospitalization. An alternative is to use the host entry receptor, angiotensin-converting enzyme 2 (ACE2), as a soluble decoy that broadly blocks SARS-associated coronaviruses with limited potential for viral escape. Here, we summarize efforts to engineer higher affinity variants of soluble ACE2 that rival the potency of affinity-matured antibodies. Strategies have also been used to increase the valency of ACE2 decoys for avid spike interactions and to improve pharmacokinetics via IgG fusions. Finally, the intrinsic catalytic activity of ACE2 for the turnover of the vasoconstrictor angiotensin II may directly address COVID-19 symptoms and protect against lung and cardiovascular injury, conferring dual mechanisms of action unachievable by monoclonal antibodies. Soluble ACE2 derivatives therefore have the potential to be next generation therapeutics for addressing the immediate needs of the current pandemic and possible future outbreaks.

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2. Specificity mutations are enriched at the base of the G-H binding loop of EFNB2, especially surrounding a phenylalanine hinge upon which the G-H loop pivots, and at a phenylalanine hook that rotates away from the EFNB2 core to engage Eph receptors. One EFNB2 mutant, D62Q, possesses pan-specificity to the attachment glycoproteins of closely related henipaviruses and has markedly diminished binding to the six Eph receptors. However, EFNB2-D62Q has high residual binding to EphB3 and EphB4. A second deep mutational scan of EFNB2 identified combinatorial mutations to further enhance specificity to NiV-G. A triple mutant of soluble EFNB2, D62Q-Q130L-V167L, has minimal binding to Eph receptors but maintains binding, albeit reduced, to NiV-G. Soluble EFNB2 decoy receptors carrying the specificity mutations were potent neutralizers of chimeric henipaviruses. These findings demonstrate how specific residue changes at the shared binding interface of a promiscuous ligand (EFNB2) can influence selectivity for multiple receptors, and may also offer insight towards the development of henipavirus therapeutics and diagnostics.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE2 2 .v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE2 2 .v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy’s therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE2 2 .v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE2 2 .v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.