Structure of a human monoclonal antibody in complex with Outer surface protein C (OspC) of the Lyme disease spirochete, Borreliella burgdorferi.

Lyme disease is a tick-borne, multisystem infection caused by the spirochete, Borreliella burgdorferi . Although antibodies have been implicated in the resolution of Lyme disease, the specific B cell epitopes targeted during human infections remain largely unknown. In this study, we characterized and defined the structural epitope of a patient-derived bactericidal monoclonal IgG ("B11") against Outer surface protein C (OspC), a homodimeric lipoprotein necessary for B. burgdorferi tick-mediated transmission and early-stage colonization of vertebrate hosts. High-resolution epitope mapping was accomplished through hydrogen deuterium exchange-mass spectrometry (HDX-MS) and X-ray crystallography. Structural analysis of B11 Fab-OspC A complexes revealed the B11 Fabs associated in a 1:1 stoichiometry with the lateral faces of OspC A homodimers such that the antibodies are essentially positioned perpendicular to the spirochete's outer surface. B11's primary contacts reside within the membrane proximal regions of α-helices 1 and 6 and adjacent loops 5 and 6 in one OspC A monomer. In addition, B11 spans the OspC A dimer interface, engaging opposing α-helix 1', α-helix 2', and loop 2-3' in the second OspC A monomer. The B11-OspC A structure is reminiscent of the recently solved mouse transmission blocking monoclonal IgG B5 in complex with OspC A , indicating a mode of engagement with OspC that is conserved across species. In conclusion, we provide the first detailed insight into the interaction between a functional human antibody and an immunodominant Lyme disease antigen long considered an important vaccine target.

Tryptanthrin Analogs Substoichiometrically Inhibit Seeded and Unseeded Tau4RD Aggregation.

Microtubule-associated protein tau is an intrinsically disordered protein (IDP) that forms characteristic fibrillar aggregates in several diseases, the most well-known of which is Alzheimer's disease (AD). Despite keen interest in disrupting or inhibiting tau aggregation to treat AD and related dementias, there are currently no FDA-approved tau-targeting drugs. This is due, in part, to the fact that tau and other IDPs do not exhibit a single well-defined conformation but instead populate a fluctuating conformational ensemble that precludes finding a stable "druggable" pocket. Despite this challenge, we previously reported the discovery of two novel families of tau ligands, including a class of aggregation inhibitors, identified through a protocol that combines molecular dynamics, structural analysis, and machine learning. Here we extend our exploration of tau druggability with the identification of tryptanthrin and its analogs as potent, substoichiometric aggregation inhibitors, with the best compounds showing potencies in the low nanomolar range even at a ~100-fold molar excess of tau4RD. Moreover, conservative changes in small molecule structure can have large impacts on inhibitory potency, demonstrating that similar structure-activity relationship (SAR) principles as used for traditional drug development also apply to tau and potentially to other IDPs.

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses.

The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing techniques to enhance the functional breadth of vaccine-elicited immune responses against the HA RBD. We present a series of "trihead" nanoparticle immunogens that display native-like closed trimeric RBDs from the HAs of several H1N1 influenza viruses. The series includes hyperglycosylated and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the receptor-binding site periphery. Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicit higher hemagglutination inhibition (HAI) and neutralizing activity than the corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation. By contrast, mosaic nanoparticle display and antigen hypervariation do not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Our results yield important insights into antibody responses against the RBD and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

Combinatorial immune refocusing within the influenza hemagglutinin head elicits cross-neutralizing antibody responses.

The head domain of influenza hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies during infection and vaccination. Here we evaluated a series of immunogens that combined several immunofocusing techniques for their ability to enhance the functional breadth of vaccine-elicited immune responses. We designed a series of "trihead" nanoparticle immunogens that display native-like closed trimeric heads from the HAs of several H1N1 influenza viruses, including hyperglycosylated variants and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the periphery of the receptor binding site (RBS). Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicited higher HAI and neutralizing activity against vaccine-matched and -mismatched H1 viruses than corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation, indicating that both of these engineering strategies contributed to improved immunogenicity. By contrast, mosaic nanoparticle display and antigen hypervariation did not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Serum competition assays and electron microscopy polyclonal epitope mapping revealed that the trihead immunogens, especially when hyperglycosylated, elicited a high proportion of antibodies targeting the RBS, as well as cross-reactive antibodies targeting a conserved epitope on the side of the head. Our results yield important insights into antibody responses against the HA head and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to complete fusion. HA is also the most variable, rapidly evolving part of influenza virus, and it is not known whether mechanisms of its activation and fusion are conserved across divergent viral subtypes. Here, we apply hydrogen-deuterium exchange mass spectrometry to compare fusion activation in two subtypes of HA, H1 and H3. Our data reveal subtype-specific behavior in the regions of HA that undergo structural rearrangement during fusion, including the fusion peptide and HA1/HA2 interface. In the presence of an antibody that inhibits the conformational change (FI6v3), we observe that acid-induced dynamic changes near the epitope are dampened, but the degree of protection at the fusion peptide is different for the two subtypes investigated. These results thus provide new insights into variation in the mechanisms of influenza HA's dynamic activation and its inhibition.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Cullin-independent recognition of HHARI substrates by a dynamic RBR catalytic domain.

RING-between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3-ubiquitin thioester intermediate prior to substrate ubiquitination. Although RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic, and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using a combination of XL-MS, HDX-MS, NMR, and biochemical studies. The results show that (1) a di-aromatic surface on the catalytic HHARI Rcat domain forms a binding platform for substrates and (2) a phosphomimetic mutation on the auto-inhibitory Ariadne domain of HHARI promotes release and reorientation of Rcat for transthiolation and substrate modification. The findings identify a direct binding interaction between a RING-between-RING ligase and its substrate and suggest a general model for RBR substrate recognition.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems.

Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein-protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

Simple Platform for Automating Decoupled LC-MS Analysis of Hydrogen/Deuterium Exchange Samples.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is capable of providing unique insight into complex biological systems that are difficult to study by other techniques. Due to arduous sample handling requirements, automating HDX experimentation for higher throughput requires specialized equipment. While recent advances have enabled automation of sample preparation and analysis, several proteins of interest and types of HDX experiments remain incompatible with automated workflows and require manual sample preparation that greatly limits experimental throughput. To expand throughput and increase the precision of HDX-MS for systems requiring manual preparation, we have developed an inexpensive autosampler capable of thawing and injecting frozen HDX-MS samples in a highly reproducible manner.

Linkage Memory in Underivatized Protonated Carbohydrates.

Carbohydrates are among the most complex class of biomolecules, and even subtle variations in their structures are attributed to diverse biological functions. Mass spectrometry has been essential for large scale glycomics and glycoproteomics studies, but the gas-phase structures and sometimes anomalous fragmentation properties of carbohydrates present long-standing challenges. Here we investigate the gas-phase properties of a panel of isomeric protonated disaccharides differing in their linkage configurations. Multiple conformations were evident for most of the structures based on their fragment ion abundances by tandem mass spectrometry, their ion mobilities in several gases, and their deuterium uptake kinetics by gas-phase hydrogen-deuterium exchange. Most notably, we find that the properties of the Y-ion fragments are characteristically influenced by the precursor carbohydrate's linkage configuration. This study reveals how protonated carbohydrate fragment ions can retain "linkage memory" that provides structural insight into their intact precursor.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8.

Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.

Rapid Differentiation of Chondroitin Sulfate Isomers by Gas-phase Hydrogen-deuterium Exchange.

Chondroitin sulfate (CS)-glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides attached to CS proteoglycans that make up a major component of biological matrices throughout both central and peripheral tissues. The position of their attached sulfate groups to the CS disaccharide is predicted to influence protein-glycan interactions and biological function. Although traditional immunohistochemical analysis of CS-GAGs in biological tissues has provided information regarding changes in GAG abundance during developmental and disease states, quantitative analysis of their specific sulfation patterns is limited due to the inherent complexity of separating CS isomers. While methods have been developed to analyze and quantify sulfation isomers using liquid phase separation, new techniques are still needed to elucidate the full biology of CS-GAGs. Here, we examine ion mobility spectrometry and gas-phase hydrogen-deuterium exchange to resolve positional sulfation isomers in the most common sulfated 4S- and 6S-CS disaccharides. The mobilities for these two isomers are highly similar and could not be resolved effectively with any drift gas tested. In contrast, gas-phase hydrogen-deuterium exchange showed very different rates of deuterium uptake with several deuterium exchange reagents, thereby presenting a promising novel and rapid approach for resolving CS isomers.

Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats.

We recently showed that perineuronal nets (PNNs) enmesh glucoregulatory neurons in the arcuate nucleus (Arc) of the mediobasal hypothalamus (MBH)1, but whether these PNNs play a role in either the pathogenesis of type 2 diabetes (T2D) or its treatment remains unclear. Here we show that PNN abundance within the Arc is markedly reduced in the Zucker diabetic fatty (ZDF) rat model of T2D, compared with normoglycaemic rats, correlating with altered PNN-associated sulfation patterns of chondroitin sulfate glycosaminoglycans in the MBH. Each of these PNN-associated changes is reversed following a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) at a dose that induces sustained diabetes remission in male ZDF rats. Combined with previous work localizing this FGF1 effect to the Arc area2-4, our finding that enzymatic digestion of Arc PNNs markedly shortens the duration of diabetes remission following icv FGF1 injection in these animals identifies these extracellular matrix structures as previously unrecognized participants in the mechanism underlying diabetes remission induced by the central action of FGF1.

Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.