Biology subjects

Chait, B. T.

Publications and source records attributed to Chait, B. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Multivalent Anti-ACE2 Nanobodies Confer Broad Pan-Sarbecovirus Protection

The continual emergence of SARS-CoV-2 variants that rapidly evade conventional spike-directed neutralizing antibodies, together with the ongoing risk of cross-species spillover and new sarbecovirus outbreaks, underscores the need to develop broadly acting, escape-resistant therapeutic agents. Here, we optimized a nanobody discovery pipeline incorporating competition-based yeast surface display assays to isolate single-chain variable heavy chain-only antibody domains (VHHs or nanobodies) that bind human ACE2 and inhibit SARS-CoV-2 entry. Dimeric VHHs, as well as bivalent and tetravalent Fc-fusion proteins exhibited markedly increased antiviral activity, blocking a broad panel of SARS-CoV-2 variants and diverse sarbecoviruses at low-nanomolar to picomolar concentrations. These agents did not affect ACE2 enzymatic function or cell surface expression. The VHH-Fc fusion proteins had favorable pharmacokinetics and conferred prophylactic protection in mouse models of both SARS-CoV-2 and SARS-CoV infection, showcasing their potential as broadly acting receptor-targeted biologics against pandemic-threat viruses.

immunology↗

bioRxiv2025

A Nature-Inspired Ion Trap for Parallel Manipulation of Ions on a Massive Scale

Parallelization has revolutionized computing and DNA sequencing but remains largely unexploited in mass spectrometry (MS), which typically analyzes ions sequentially. We introduce a nature-inspired ion trap (MultiQ-IT) that enables massively parallel MS. The device comprises a cubic array of small quadrupoles forming multiple ion entry and exit ports, allowing >10 ions to be confined and manipulated simultaneously. This architecture enables selective depletion of singly charged ions in real time, greatly improving signal-to-noise ratios and detection sensitivity. The trap also functions as a parallel ion splitter, transmitting ions into multiple m/z-specific beams. We demonstrate scalable ion throughput, real-time charge discrimination, and parallel beam separation, suggesting a path toward truly parallel MS. Our results offer a foundation for next-generation, high-throughput proteomic and metabolomic analyses.

biophysics↗

bioRxiv2024

RapA opens the RNA polymerase clamp to disrupt post-termination complexes and prevent cytotoxic R-loop formation

Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the Swi2/Snf2 ATPase RapA. Here, we determined PTC structures on negatively-supercoiled DNA as well as of RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. We show that RapA helps control cytotoxic R-loop formation in vivo, likely by disrupting PTCs. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We suggest that analagous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold significance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability.

biophysics↗

bioRxiv2024

The Molecular Architecture of the Nuclear Basket

The nuclear pore complex (NPC) is the sole mediator of nucle-ocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPCs structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of Nups in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport

biophysics↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2023

Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.

Nanobodies are single domain antibody variants that bind an antigen with the precision and affinity of a conventional antibody at only a fraction of their size. In solving the crystal structures of our nanobody-GFP complexes and compared with other available structures, we uncover mechanism that enable nanobodies to function so efficiently and effectively as single-domain antibodies. We show that unlike conventional antibodies, a nanobody repertoire maximizes sampling of their antigen surface by binding a single antigen in at least three different orientations which can be predicted by their paratope composition. We also structurally reengineering these nanobodies to improve their antigen affinity, their stability, or both - results which also revealed the strong connection between nanobody stability and affinity. We achieved this by either directly modifying the paratope, or by altering a particular region within their third framework, which is a highly conserved area that we determined plays a role in controlling nanobody stability. Our study suggests that these unique characteristics of nanobodies allow them to interact with antigens as effectively as conventional antibodies, despite their smaller size. This understanding provides methods to facilitate optimizing, humanizing and functionalizing nanobodies, thus paving the way for their utilization in diverse areas such as research, diagnostics, and therapeutic development. Significance StatementNanobodies are a unique type of antibody fragment found in select animals, containing all its antigen binding ability reduced to a single [~]15 kDa protein. There is increasing development of nanobodies for research, diagnostics, and therapeutics, yet how nanobodies function so effectively as single domain antigen binders with the precision and affinity of conventional antibodies is unclear. In this study, we present key observations to help answer this question, where one key finding is the strong relationship between nanobody stability and antigen affinity aided by the identification of a highly conserved region in nanobodies essential for maintaining nanobody stability. This region may have been retained in nanobodies in lieu of stabilizing mechanisms induced by dimerization as seen in conventional antibodies.

biochemistry↗

bioRxiv2023

Using a function-first "scout fragment"-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic scout fragments, which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

biochemistry↗

bioRxiv2023

Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast, Fridy et al. 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.

biochemistry↗

bioRxiv2023

Differential dynamics specify MeCP2 function at methylated DNA and nucleosomes

Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a leading cause of monogenic intellectual disabilities in females. Despite its significant biomedical relevance, the mechanism by which MeCP2 navigates the chromatin epigenetic landscape to regulate chromatin structure and gene expression remains unclear. Here, we used correlative single-molecule fluorescence and force microscopy to directly visualize the distribution and dynamics of MeCP2 on a variety of DNA and chromatin substrates. We found that MeCP2 exhibits differential diffusion dynamics when bound to unmethylated and methylated bare DNA. Moreover, we discovered that MeCP2 preferentially binds nucleosomes within the context of chromatinized DNA and stabilizes them from mechanical perturbation. The distinct behaviors of MeCP2 at bare DNA and nucleosomes also specify its ability to recruit TBLR1, a core component of the NCoR1/2 co-repressor complex. We further examined several RTT mutations and found that they disrupt different aspects of the MeCP2-chromatin interaction, rationalizing the heterogeneous nature of the disease. Our work reveals the biophysical basis for MeCP2s methylation-dependent activities and suggests a nucleosome-centric model for its genomic distribution and gene repressive functions. These insights provide a framework for delineating the multifaceted functions of MeCP2 and aid in our understanding of the molecular mechanisms of RTT.

biophysics↗

bioRxiv2023

Dynamic molecular mechanism of the nuclear pore complex permeability barrier

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

biophysics↗

bioRxiv2023

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring. Statement of SignificanceLINE-1 ORF1p transposon protein is pervasively expressed in many cancers and a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 L plasma are novel, rapid, cost-effective tools in cancer detection and monitoring.

cancer biology↗

bioRxiv2022

Mex67 paralogs mediate division of labor in trypanosome RNA processing and export.

In opistokhonts (animals and fungi), mRNA export to the cytoplasm is mediated by the Mex67/Mtr2 (NXF1/NXT1) heterodimer via the nuclear pore complex (NPC). In contrast to most nucleocytoplasmic transport, mRNA export requires ATP-dependent remodeling machinery, and in animals and fungi is Ran-independent. While most eukaryotes possess one Mex67 gene, trypanosomes have three distinct Mex67 paralogs, while retaining a single Mtr2 gene. We show here that these paralogs, TbMex67, TbMex67b and TbMex67L, have differing and non-redundant roles in RNA export. Specifically, TbMex67 and TbMex67b retain a canonical role in mRNA export, albeit associating with specific mRNA cohorts, but in contrast, TbMex67L is primarily involved in ribosome biogenesis. Together with the association of all Mex67 paralogs with the Ran machinery, these findings indicate significant departures in RNA export mechanisms in these divergent organisms, with implications for evolutionary origins and diversity in control of gene expression.

cell biology↗

bioRxiv2022

Structural basis for substrate selection by the SARS-CoV-2 replicase

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP3,4. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5 RNA cap6.

biophysics↗

bioRxiv2022

Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity

Human shelterin is a six-subunit complex - comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1 - that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinantly produced components. By combining negative-stain electron microscopy (EM), crosslinking mass spectrometry (XLMS), mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they are extensively conformationally heterogenous. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 OB fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Both shelterin and the TRF1/TIN2/TPP1/POT1 subcomplex primarily adopt fully dimeric complexes, even in the absence of DNA substrates. TRF1/TIN2/TPP1/POT1 and shelterin complex showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

biochemistry↗

bioRxiv2021

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

molecular biology↗

bioRxiv2021

The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying MIDAS-UBL switching between strongly- and weakly-bound states is unknown. Here, we use single-molecule optical tweezers to investigate the force-dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to tenfold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1s MIDAS domain forms catch bonds with more than one UBL-substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

biophysics↗

bioRxiv2021

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

biochemistry↗

bioRxiv2021

Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein crosslinking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3-segment of the product-RNA generated by backtracking extrudes through the RdRp NTP-entry tunnel, that a mismatched nucleotide at the product-RNA 3-end frays and enters the NTP-entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals. Significance StatementThe COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome is replicated and transcribed by its RNA-dependent RNA polymerase (RdRp), which is the target for antivirals such as remdesivir. We use a combination of approaches to show that backtracking (backwards motion of the RdRp on the template RNA) is a feature of SARS-CoV-2 replication/transcription. Backtracking may play a critical role in proofreading, a crucial process for SARS-CoV-2 resistance against many antivirals.

biochemistry↗