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Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.

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.

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 silicomutagenesis 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.

A potential therapeutic strategy for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding to the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant variant, named sACE22.v.2.4, that exhibits subnanomolar affinity to the receptor-binding domain (RBD) of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identify other ACE2 variants that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified multiple ACE2 double mutants that bind SARS-CoV-2 S with tighter affinity as compared to the wild type, most notably L79V;N90D that binds RBD similarly to ACE22.v.2.4. The experimental validation of the double mutants successfully demonstrates the use of machine learning approaches for engineering protein–protein interactions and identifying high-affinity ACE2 peptides for targeting SARS-CoV-2.

Ion channels are often modulated by intracellular calcium levels. TRPV1, a channel responsible for the burning pain sensation in response to heat, acid or capsaicin, is desensitized at high intracellular calcium concentrations. We recently identified a multiligand-binding site in the N-terminal ankyrin repeat domain (ARD) of TRPV1 that binds ATP and sensitizes the channel. Calcium-calmodulin binds the same site and is necessary for calcium-mediated TRPV1 desensitization. Here, we examine in more detail the conservation of this TRPV1 multiligand-binding site in other species. Furthermore, using sequence analysis, we determine that the unusually twisted shape of the TRPV1-ARD is likely conserved in other TRPV channels, but not in the ARDs of other TRP subfamilies.

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy (cryo-EM) structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states provided a glimpse of functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to various 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 novel 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.

Introduction:Vaccine hesitancy and the continuous emergence of SARS-CoV-2 variants of concern that partially escape vaccine-induced immune responses highlight the urgent need for the development of effective therapeutics for COVID-19 patients.Hypothesis:The Engineered high-affinity ACE2 peptide has a therapeutic effect for SARS-CoV-2 and its variant-induced acute lung vascular injury.Methods:K18-hACE2 transgenic mice were inoculated with SARS-CoV-2 WA-1/2020 or P.1 variant of concerns to induce acute lung injury. The sACE22.v2.4-IgG1 was injected once a day for consecutively 7 days starting from 12 hours at 10 mg/kg/day or 24 hours at 15 mg/kg/day post-SARS-CoV-2 inoculation.Results:We demonstrated that the higher binding affinity of sACE22.v2.4-IgG1 to S proteins of the original SARS-CoV-2 WA-1/2020 isolate and the B.1.1.7 (Alpha, UK isolate), B.1.351 (Beta, South Africa isolate), P.1 (Gamma, Brazil isolate), and B.1.617.2 (Delta, India isolate) SARS-CoV-2 variants of concern compared to wild-type soluble ACE2. Using humanized K18-hACE2 mice, we found that both SARS-CoV-2 isolate WA-1/2020 and the P.1 variant of concern induced severe acute lung endothelial injury, which resulted in the severe transvascular leak, lung edema, and death. The P.1 variant induced severe lung vascular leak and death at an earlier time point at Day 5 compared to WA-1 at Day 7, consistent with increased viral entry and replication. Treatment with sACE22.v2.4-IgG1 decoy peptide could be initiated as late as 24h after inoculation with a lethal dose of the SARS-CoV-2 WA-1/2020 isolate and profoundly reduces lung vascular injury and mortality. For the P.1 variant of concern, ACE22.v2.4-IgG1 decoy peptide markedly improved survival and reduced lung vascular injury if administered early at 12 hours post-inoculation.Conclusion:sACE22.v2.4-IgG1 has an exceptional therapeutic efficacy to SARS-CoV-2 WA-1/2020 and variant P.1 -induced acute lung vascular injury.