Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli.

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

Anti-Immune Complex Antibodies are Elicited During Repeated Immunization with HIV Env Immunogens.

Vaccination strategies against HIV-1 aim to elicit broadly neutralizing antibodies (bnAbs) using prime-boost regimens with HIV envelope (Env) immunogens. Early antibody responses to easily accessible epitopes on these antigens are directed to non-neutralizing epitopes instead of bnAb epitopes. Autologous neutralizing antibody responses appear upon boosting once immunodominant epitopes are saturated. Here we report another type of antibody response that arises after repeated immunizations with HIV Env immunogens and present the structures of six anti-immune complexes discovered using polyclonal epitope mapping. The anti-immune complex antibodies target idiotopes composed of framework regions of antibodies bound to Env. This work sheds light on current vaccine development efforts for HIV, as well as for other pathogens, in which repeated exposure to antigen is required.

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows.

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines.

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

Effect of verbal instructions on trunk muscle activity during volitional preemptive abdominal contraction.

OBJECTIVE: This study investigated the effect of Verbal Instruction (VI) strategies on trunk muscle contraction among healthy subjects. The effect of three VI Abdominal Drawing-In Maneuver (ADIM) and two VI Abdominal Bracing Maneuver (ABM) strategies on left Internal (LIO) and External Oblique (LEO) and bilateral superficial Multifidi (sMf) activation was examined. DESIGN: Within-subjects, repeated measure design. METHODS: Surface EMG (sEMG) measured LIO, LEO, and sMf activity in 28 subjects (mean age 23.5 ± 5.5 years). Testing included five supine hook-lying and five quiet standing conditions. RESULTS: One-way ANOVAs demonstrated no significant main effect for ADIM or ABM in supine or standing (p > .05). Muscle activation amplitudes during VPAC conditions demonstrated higher mean values for standing versus supine (p < .05) except for two conditions involving LEO. Friedman Tests for dominant strategy demonstrated a significant main effect for ADIM-VI and ABM-VI strategies. Post-hoc testing generally showed the dominant strategy to be significantly higher versus others. CONCLUSION: No single preferred VI cue for ADIM or ABM was observed. Each subject's dominant strategy dictated the most suitable VI. Standing was preferred for LIO and sMf activation, whereas position did not change LEO activation. Non-significant correlations between all muscle pairings during all ADIM and ABM strategies were observed. These findings may suggest the need for healthcare providers who understand the intricacies of trunk stability to teach and monitor VPAC with either ADIM or ABM options.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli.

The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension1. Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e., they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). In an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization2. Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.

Structure of mechanically activated ion channel OSCA2.3 reveals mobile elements in the transmembrane domain.

Members of the OSCA/TMEM63 family are mechanically activated ion channels and structures of some OSCA members have revealed the architecture of these channels and structural features that are potentially involved in mechanosensation. However, these structures are all in a similar state and information about the motion of different elements of the structure is limited, preventing a deeper understanding of how these channels work. Here, we used cryoelectron microscopy to determine high-resolution structures of Arabidopsis thaliana OSCA1.2 and OSCA2.3 in peptidiscs. The structure of OSCA1.2 matches previous structures of the same protein in different environments. Yet, in OSCA2.3, the TM6a-TM7 linker adopts a different conformation that constricts the pore on its cytoplasmic side. Furthermore, coevolutionary sequence analysis uncovered a conserved interaction between the TM6a-TM7 linker and the beam-like domain (BLD). Our results reveal conformational heterogeneity and differences in conserved interactions between the TMD and BLD among members of the OSCA family.

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples is a challenging goal that would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices, we call DNA nanoswitch calipers, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement with previously reported structures.

Real-world utilization of guideline-directed genetic testing in inherited cardiovascular diseases.

Background: Cardiovascular disease continues to be the leading cause of death globally. Clinical practice guidelines aimed at improving disease management and positively impacting major cardiac adverse events recommend genetic testing for inherited cardiovascular conditions such as dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), long QT syndrome (LQTS), hereditary amyloidosis, and familial hypercholesterolemia (FH); however, little is known about how consistently practitioners order genetic testing for these conditions in routine clinical practice. This study aimed to assess the adoption of guideline-directed genetic testing for patients diagnosed with DCM, HCM, LQTS, hereditary amyloidosis, or FH. Methods: This retrospective cohort study captured real-world evidence of genetic testing from ICD-9-CM and ICD-10-CM codes, procedure codes, and structured text fields of de-identified patient records in the Veradigm Health Insights Ambulatory EHR Research Database linked with insurance claims data. Data analysis was conducted using an automated electronic health record analysis engine. Patient records in the Veradigm database were sourced from more than 250,000 clinicians serving over 170 million patients in outpatient primary care and specialty practice settings in the United States and linked insurance claims data from public and private insurance providers. The primary outcome measure was evidence of genetic testing within six months of condition diagnosis. Results: Between January 1, 2017, and December 31, 2021, 224,641 patients were newly diagnosed with DCM, HCM, LQTS, hereditary amyloidosis, or FH and included in this study. Substantial genetic testing care gaps were identified. Only a small percentage of patients newly diagnosed with DCM (827/101,919; 0.8%), HCM (253/15,507; 1.6%), LQTS (650/56,539; 1.2%), hereditary amyloidosis (62/1,026; 6.0%), or FH (718/49,650; 1.5%) received genetic testing. Conclusions: Genetic testing is underutilized across multiple inherited cardiovascular conditions. This real-world data analysis provides insights into the delivery of genomic healthcare in the United States and suggests genetic testing guidelines are rarely followed in practice.

Germline-targeting SOSIP trimer immunization elicits precursor CD4 binding-site targeting broadly neutralizing antibodies in infant macaques.

A vaccine that can achieve protective immunity prior to sexual debut is critical to prevent the estimated 410,000 new HIV infections that occur yearly in adolescents. As children living with HIV can make broadly neutralizing antibody (bnAb) responses in plasma at a faster rate than adults, early childhood is an opportune window for implementation of a multi-dose HIV immunization strategy to elicit protective immunity prior to adolescence. Therefore, the goal of our study was to assess the ability of a B cell lineage-designed HIV envelope SOSIP to induce bnAbs in early life. Infant rhesus macaques (RMs) received either BG505 SOSIP or the germline-targeting BG505 GT1.1 SOSIP (n=5/group) with the 3M-052-SE adjuvant at 0, 6, and 12 weeks of age. All infant RMs were then boosted with the BG505 SOSIP at weeks 26, 52 and 78, mimicking a pediatric immunization schedule of multiple vaccine boosts within the first two years of life. Both immunization strategies induced durable, high magnitude binding antibodies and plasma autologous virus neutralization that primarily targeted the CD4-binding site (CD4bs) or C3/465 epitope. Notably, three BG505 GT1.1-immunized infants exhibited a plasma HIV neutralization signature reflective of VRC01-like CD4bs bnAb precursor development and heterologous virus neutralization. Finally, infant RMs developed precursor bnAb responses at a similar frequency to that of adult RMs receiving a similar immunization strategy. Thus, a multi-dose immunization regimen with bnAb lineage designed SOSIPs is a promising strategy for inducing protective HIV bnAb responses in childhood prior to adolescence when sexual HIV exposure risk begins.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1-envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is measured more often than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy of active or passive immunization and the therapeutic outcome of the latter. Many NAbs neutralize HIV-1 CZA97.012, a clone of a Clade-C isolate, to ~100%. But here NAb PGT151, directed to a fusion-peptide epitope, left a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, left no detectable persistent fraction. The divergence in persistent fractions was further analyzed by depletion of pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased, whereas neutralization by the depleting NAb decreased. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-ß19 transition in a defect of the glycan shield on CZA97.012 Env. NAb binding to affinity-fractionated soluble native-like CZA97.012 trimer differed commensurately with neutralization in analyses by ELISA and surface plasmon resonance. Glycan differences between PGT151- and PGT145-purified trimer fractions were then demonstrated by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-Å resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. The trimer adopted a closed conformation, refuting apex opening as the cause of reduced PGT145 binding to the PGT151-purified form. The evidence suggests that differences in binding and neutralization after trimer purification or pseudovirus depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants affect antigenicity through direct effects on antibody contacts, whereas others act allosterically.

Structure and Dynamics Guiding Design of Antibody Therapeutics and Vaccines.

Antibodies and other new antibody-like formats have emerged as one of the most rapidly growing classes of biotherapeutic proteins. Understanding the structural features that drive antibody function and, consequently, their molecular recognition is critical for engineering antibodies. Here, we present the structural architecture of conventional IgG antibodies alongside other formats. We emphasize the importance of considering antibodies as conformational ensembles in solution instead of focusing on single-static structures because their functions and properties are strongly governed by their dynamic nature. Thus, in this review, we provide an overview of the unique structural and dynamic characteristics of antibodies with respect to their antigen recognition, biophysical properties, and effector functions. We highlight the numerous technical advances in antibody structure prediction and design, enabled by the vast number of experimentally determined high-quality structures recorded with cryo-EM, NMR, and X-ray crystallography. Lastly, we assess antibody and vaccine design strategies in the context of structure and dynamics.