Biologics, including monoclonal antibodies (mAbs), are widely used therapeutics due to their high specificity, yet off-target interactions remain an underappreciated risk for safety and efficacy. To systematically assess antibody specificity, we applied rapid extracellular antigen profiling (REAP) to evaluate 174 FDA-approved and clinical-stage antibodies against 6,172 human extracellular proteins. We found a substantial burden of off-target reactivity, with 28% of antibodies exhibiting at least one off-target hit. Structural and biophysical analyses revealed that off-target binding arises from antibody intrinsic properties and epitope mimicry, either within related protein families or across structurally unrelated proteins. We further identified new off-target interactions of tanezumab and engineered its variable domains to eliminate off-target binding while preserving target affinity and developability. These findings highlight the prevalence of specific off-target reactivity in therapeutic antibodies and underscore the importance of evaluating specificity early in biologic drug development.

The myriad microorganisms that live in close association with humans have diverse effects on physiology, yet the molecular bases for these impacts remain mostly unknown(1-3). Classical pathogens often invade host tissues and modulate immune responses through interactions with human extracellular and secreted proteins (the 'exoproteome'). Commensal microorganisms may also facilitate niche colonization and shape host biology by engaging host exoproteins; however, direct exoproteome-microbiota interactions remain largely unexplored. Here we developed and validated a novel technology, BASEHIT, that enables proteome-scale assessment of human exoproteome-microbiome interactions. Using BASEHIT, we interrogated more than 1.7 million potential interactions between 519 human-associated bacterial strains from diverse phylogenies and tissues of origin and 3,324 human exoproteins. The resulting interactome revealed an extensive network of transkingdom connectivity consisting of thousands of previously undescribed host-microorganism interactions involving 383 strains and 651 host proteins. Specific binding patterns within this network implied underlying biological logic; for example, conspecific strains exhibited shared exoprotein-binding patterns, and individual tissue isolates uniquely bound tissue-specific exoproteins. Furthermore, we observed dozens of unique and often strain-specific interactions with potential roles in niche colonization, tissue remodelling and immunomodulation, and found that strains with differing host interaction profiles had divergent interactions with host cells in vitro and effects on the host immune system in vivo. Overall, these studies expose a previously unexplored landscape of molecular-level host-microbiota interactions that may underlie causal effects of indigenous microorganisms on human health and disease.

The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.

The cytokine IL-18 has immunostimulatory effects but is negatively regulated by a secreted binding protein, IL-18BP, that limits IL-18's anticancer efficacy. A decoy-resistant form of IL-18 (DR-18) that avoids sequestration by IL-18BP while maintaining its immunostimulatory potential has recently been developed. Here, we investigated the therapeutic potential of DR-18 in renal cell carcinoma (RCC). Using pantumor transcriptomic data, we found that clear cell RCC had among the highest expression of IL-18 receptor subunits and IL18BP of tumor types in the database. In samples from patients with RCC treated with immune checkpoint inhibitors, IL-18BP protein expression increased in the tumor microenvironment and in circulation within plasma in nonresponding patients, and it decreased in the majority of responding patients. We used immunocompetent RCC murine models to assess the efficacy of DR-18 in combination with single- and dual-agent anti-PD-1 and anti-CTLA-4. In contrast to preclinical models of other tumor types, in RCC models, DR-18 enhanced the activity of anti-CTLA-4 but not anti-PD-1 treatment. This activity correlated with intratumoral enrichment and clonal expansion of effector CD8+ T cells, decreased Treg levels, and enrichment of proinflammatory antitumor myeloid cell populations. Our findings support further clinical investigation of the combination of DR-18 and anti-CTLA-4 in RCC.

Interleukin-18 (IL-18) promotes natural killer (NK) and T cell production of interferon (IFN)-γ, a key factor in resistance to Toxoplasma gondii, but previous work has shown a limited role for endogenous IL-18 in control of this parasite. Although infection with T. gondii results in release of IL-18, the production of IFN-γ induces high levels of the IL-18 binding protein (IL-18BP). Antagonism of IL-18BP with a “decoy-to-the-decoy” (D2D) IL-18 construct that does not signal but rather binds IL-18BP results in enhanced innate lymphoid cell (ILC) and T cell responses and improved parasite control. In addition, the use of IL-18 resistant to IL-18BP (“decoy-resistant” IL-18 [DR-18]) is more effective than exogenous IL-18 at promoting innate resistance to infection. DR-18 enhances CD4+ T cell production of IFN-γ but results in CD4+ T cell-mediated pathology. Thus, endogenous IL-18BP restrains aberrant immune pathology, and this study highlights strategies that can be used to tune this regulatory pathway for optimal anti-pathogen responses.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Abstract Background Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials. Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma. Results In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4. Conclusions In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.

Background Cytokine-based drugs are being explored as alternative cancer immunotherapies.1 While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18's therapeutic efficacy.2–5 A modified version of IL-18, termed 'decoy-resistant' or DR-18, that can avoid trapping by IL-18BP while maintaining immune signaling potential, has recently been developed.6 Here, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC). Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In Renca, plasma was collected post-treatment and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed, and immune cell depletion studies were conducted. Results In Renca, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival (figure 1). The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model produced similar results, showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to controls (figure 2). Single-cell transcriptomics demonstrated changes in intra-tumoral T cells, macrophages, and granulocytes with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling (figure 3). Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma as equally required for efficacy of DR-18 plus anti-CTLA-4 (figure 4). Conclusions We identify DR-18, a 'decoy-resistant' IL-18, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Investigation is ongoing to further elucidate the cellular mechanism of action of this regimen and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. References Conlon KC, MD Miljkovic, TA Waldmann. Cytokines in the Treatment of Cancer. J Interferon Cytokine Res, 2019;39(1):6–21. Guo L, IS Junttila, WE Paul. Cytokine-induced cytokine production by conventional and innate lymphoid cells. Trends Immunol, 2012;33(12):598–606. Robertson MJ, et al. Clinical and biological effects of recombinant human interleukin-18 administered by intravenous infusion to patients with advanced cancer. Clin Cancer Res, 2006;12(14 Pt 1):4265–73. Tarhini AA, et al. A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer, 2009;115(4):859–68. Dinarello CA, et al. Interleukin-18 and IL-18 binding protein. Front Immunol, 2013;4:289. Zhou T, et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature, 2020;583(7817):609–614. Ethics Approval This study was approved by the institutional IACUC (protocol #2023–20152) and all institutional guidelines were followed.

Some hematological malignancies such as multiple myeloma are inherently resistant to immune-mediated antitumor responses, the cause of which remains unknown. Allogeneic bone marrow transplantation (alloBMT) is the only curative immunotherapy for hematological malignancies due to profound graft-versus-tumor (GVT) effects, but relapse remains the major cause of death. We developed murine models of alloBMT where the hematological malignancy is either sensitive [acute myeloid leukemia (AML)] or resistant (myeloma) to GVT effects. We found that CD8 + T cell exhaustion in bone marrow was primarily alloantigen-driven, with expression of inhibitory ligands present on myeloma but not AML. Because of this tumor-independent exhaustion signature, immune checkpoint inhibition (ICI) in myeloma exacerbated graft-versus-host disease (GVHD) without promoting GVT effects. Administration of post-transplant cyclophosphamide (PT-Cy) depleted donor T cells with an exhausted phenotype and spared T cells displaying a stem-like memory phenotype with chromatin accessibility present in cytokine signaling genes, including the interleukin-18 (IL-18) receptor. Whereas ICI with anti–PD-1 or anti–TIM-3 remained ineffective after PT-Cy, administration of a decoy-resistant IL-18 (DR-18) strongly enhanced GVT effects in both myeloma and leukemia models, without exacerbation of GVHD. We thus defined mechanisms of resistance to T cell–mediated antitumor effects after alloBMT and described an immunotherapy approach targeting stem-like memory T cells to enhance antitumor immunity.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Infection with SARS-CoV-2 results in a broad spectrum of immunological and clinical outcomes for patients with COVID-19. While dysregulated innate immunity has been extensively explored in severe disease, autoantibody responses and their impact on disease trajectories remain uncharted. Here, we enrolled a cohort of 194 SARS-CoV-2 infected patients and uninfected individuals, and employed a high-throughput autoantibody discovery platform, Rapid Extracellular Antigen Profiling (REAP), to assess the prevalence of autoantibodies against 2,770 extracellular and secreted proteins in COVID-19 disease. We found that COVID-19 patients harbor dramatically expanded autoantibody repertoires compared to uninfected controls. We further established that immunomodulatory proteins including cytokines, chemokines, complements, and cell surface receptors, are frequent targets of autoantibodies. Longitudinal analysis revealed the existence of both pre-existing autoantibodies, as well as a broad subset of autoantibodies induced upon SARS-CoV-2 infection. We uncovered distinct mechanisms by which immune-targeting autoantibodies perturb immunological functions, including interference of immunoreceptor signaling, depletion of circulating leukocytes, and modulation of antiviral antibody responses. Concordantly, murine surrogates of these autoantibodies hinder immune activation and exacerbate disease severity in a mouse model of SARS-CoV-2 infection. In summary, through the lens of an unbiased proteome-scale survey for autoantibody targets, these findings implicate humoral immunopathology as an integral aspect of COVID-19 pathogenesis with diverse impacts on immune functionality and clinical outcomes.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

Serological assays can detect anti-SARS-CoV-2 (SARS2) antibodies, but their sensitivity often comes at the expense of specificity. Here we used a Ternary Automated Blood Im-munoassay (TRABI) to assess the IgG response against SARS2 in 3,815 prepandemic plasma samples and 126 virologically and/or clinically confirmed COVID-19 samples. Posterior probabilities were calculated from 3x8 measurements of logarithmically diluted samples against the ectodomain and the receptor-binding domain of the spike protein and the nucleoprotein. We then performed 429,624 assays on 17,901 blood samples from patients of the University Hospital Zurich and from healthy blood donors. We found se-ropositivity in 44 of 8,591 patients and in 26 of 5,388 blood donors from December 2019 to May 2020. Western blotting confirmed seropositivity in COVID samples but in none of the prepandemic samples. Solution-equilibrium measurements revealed immunodominant antibodies with nanomolar affinity in COVID samples, whereas prepandemic plasma showed lower affinities despite similar titers for individual SARS2 antigens. Hence, TRABI identifies seropositive individuals in large unselected cohorts, discriminates be-tween SARS2 immunity and low-affinity crossreactivity, and is therefore suitable for large-scale nationwide screening campaigns.

We describe the evolution of severe acute respiratory coronavirus 2 (SARS-CoV-2) seroprevalence in the greater area of Zurich, Switzerland, a region that has been only mildly hit by the pandemic in spite of hosting an international airport hub and a highly mobile population. Seroprevalence studies in low-prevalence settings require large sample sizes and high-specificity methodologies. To address this particular challenge, we developed a Tripartite Automated Blood Immunoassay to assess the IgG response against three SARS-CoV-2 proteins on approximately 409000 samples from university hospital patients and healthy blood donors. The seroprevalence increased in March 2020 (0.3%; CI95%: 0.1% - 0.5%) in the cohort of the hospital patients but rapidly plateaued in April at 1.1-1.3%, with a slight drop in June, then decreased in July to 0.3-0.7%. Seropositive samples were confirmed with Western Blotting and liquid-phase binding assays. Employing a dynamic transmission model that describes SARS-CoV-2 transmission and seroconversion in the general population of the Canton of Zurich, we estimated an infection fatality ratio of 0.6% (CI95%: 0.4%-0.8%). We conclude that a small proportion of the population in the greater area of Zurich has been exposed to SARS-CoV-2, with an IFR that is similar to that of other European areas. The evolution of seroprevalence points to a high effectiveness of containment measures and/or to rapid loss of humoral responses.

Serological assays can detect anti-SARS-CoV-2 (SARS2) antibodies, but their sensitivity often comes at the expense of specificity. Here we developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against SARS2. Calibration was per-formed with 90 prepandemic and 55 virologically and clinically confirmed COVID-19 sam-ples. Posterior probabilities of seropositivities were calculated from 3x8 measurements of logarithmically diluted samples against the ectodomain and the receptor-binding domain of the spike protein and the nucleoprotein. We then performed 760'320 assays on 5'503 prepandemic and 26'177 copandemic samples from hospital patients and healthy blood donors. We found 176 seropositive samples between December 2019 and May 2020. The seroprevalence increased conspicuously in March 2020 but plateaued in late April at 0.8-1.6% in both cohorts, indicating an equilibrium between new infections and the waning of immunity. This points to a high effectiveness of containment measures and/or to unex-pectedly rapid loss of humoral responses.

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5′N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5′ moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Hsp90α and Hsp90β are implicated in a number of cancers and neurodegenerative disorders but the lack of selective pharmacological probes confounds efforts to identify their individual roles. Here, we analyzed the binding of an Hsp90α-selective PU compound, PU-11-trans, to the two cytosolic paralogs. We determined the co-crystal structures of Hsp90α and Hsp90β bound to PU-11-trans, as well as the structure of the apo Hsp90β NTD. The two inhibitor-bound structures reveal that Ser52, a nonconserved residue in the ATP binding pocket in Hsp90α, provides additional stability to PU-11-trans through a water-mediated hydrogen-bonding network. Mutation of Ser52 to alanine, as found in Hsp90β, alters the dissociation constant of Hsp90α for PU-11-trans to match that of Hsp90β. Our results provide a structural explanation for the binding preference of PU inhibitors for Hsp90α and demonstrate that the single nonconserved residue in the ATP-binding pocket may be exploited for α/β selectivity.

Hsp90 chaperones undergo ATP-driven conformational changes during the maturation of client proteins, populating a closed state upon ATP binding in which the N-terminal domains of the homodimer form a second inter-protomer dimer interface. A structure of GRP94, the endoplasmic reticulum hsp90, in a closed conformation has not been described, and the determinants that regulate closure are not well understood. Here, we determined the 2.6-Å structure of AMPPNP-bound GRP94 in the closed dimer conformation. The structure includes the pre-N domain, a region preceding the N-terminal domain that is highly conserved in GRP94, but not in other hsp90s. We show that the GRP94 pre-N domain is essential for client maturation, and we identify the pre-N domain as an important regulator of ATPase rates and dimer closure. The structure also reveals a GRP94:polypeptide interaction that partially mimics a client-bound state. The results provide structural insight into the ATP-dependent client maturation process of GRP94.

DNA binding proteins search for specific targets amidst an excess of non-target DNA largely through facilitated diffusion. While DNA is crowded with proteins in vivo, little is known about how protein roadblocks affect diffusion. Here, we use single-molecule fluorescence imaging and DNA curtains generated by nano-fabricated chromium arrays to investigate how Msh2-Msh3, a eukaryotic mismatch repair (MMR) complex, navigates on crowded DNA. We discovered that Msh2-Msh3, in contrast to the homologous protein complex Msh2-Msh6, diffuses on DNA by hopping. Furthermore, hopping allows Msh2-Msh3 to bypass protein roadblocks, including nucleosomes. Despite hopping over roadblocks, we observed that Msh2-Msh3 maintains sufficient contact with DNA to bind single DNA lesions. Remarkably, the primary Msh3 DNA binding domain, the mispair binding domain (MBD), translates the ability to hop to a Msh2-Msh6(3-MBD) chimera. This work provides a model for how Msh2-Msh3 locates lesions outside of replication-coupled MMR, provides insight into the structural pre-requisites for protein hopping, and shows how hopping may accelerate target search in the context of chromatin.

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.