Bacterial fimbrial adhesins such as FimH are critical for host colonization and persistence under the mechanical forces encountered at sites of infection such as the urinary tract. The molecular mechanisms by which FimH, a key virulence factor of uropathogenic Escherichia coli, regulates its binding to host cell surface mannose moieties through conformational switching remain incompletely understood. FimH operates across a range of conformations that includes low- (LAS), intermediate-, and high-affinity (HAS) states and forms catch bonds that paradoxically strengthen under force. The allosteric pathways governing these transitions remain poorly defined due to experimental limitations that restrict understanding of key dynamic phenomena that underlie ligand-triggered conformational shifts and force-induced long-lived interactions. Such understanding is central to drug discovery efforts to target bacterial adhesion. Here, we present a model system that fully recapitulates the conformational repertoire of FimH in the absence of its pilin domain. Our findings demonstrate that a single mutation in the lectin domain stabilizes the LAS while allowing for ligand-binding-induced transition to a HAS-like conformation and catch bond formation, mirroring the behavior of the native FimH adhesin. We propose a dynamic allosteric mechanism that involves ultraslow, low-frequency dynamics for the ability of FimH to sustain long-lived interactions with mannose, under both static and force conditions.

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii, respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing murine models of uncomplicated and catheter-associated UTIs in vivo.

A long-standing limitation of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) has been the difficulty in accurately measuring amide exchange with single amide resolution. Excitation of peptides or proteins during ionization, ion transmission, or collisional activation rapidly induces intermolecular hydrogen migration, leading to a loss of the deuterium-labeled state; a term commonly known as "scrambling." Electron-based fragmentation methods in conjunction with gentle ion transmission settings can minimize scrambling but often not completely. Levels of scrambling have been shown to vary with ion transmission settings, peptide charge, and size, but the general properties that govern the susceptibility of peptides to scrambling are not well understood. Furthermore, it remains unclear whether scrambling is generally a global process or if local scrambling networks commonly exist within peptides. Here, we examine a panel of peptides using gentle electron transfer dissociation and map the activation thresholds of scrambling to define a relationship between peptide charge density and scrambling propensity. This study suggests that by and large, the scrambling process has a single activation threshold and involves all exchangeable sites within a peptide. For some peptides, the activation energy required for scrambling is surprisingly close to that of amide bond dissociation.

Previously, we reconstituted a minimal functional kinetochore from recombinant Saccharomyces cerevisiae proteins that was capable of transmitting force from dynamic microtubules to nucleosomes containing the centromere-specific histone variant Cse4 (Hamilton et al. 2020). This work revealed two paths of force transmission through the inner kinetochore: through Mif2 and through the Okp1/Ame1 complex (OA). Here, using a chimeric DNA sequence that contains crucial centromere-determining elements of the budding yeast point centromere, we demonstrate that the presence of centromeric DNA sequences in Cse4-containing nucleosomes significantly strengthens OA-mediated linkages. Our findings indicate that centromeric sequences are important for the transmission of microtubule-based forces to the chromosome.

An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.

We present pyHXExpress, a customizable codebase for automated high-throughput multimodal analysis of all spectra generated from HDX-MS experiments. The workflow was validated against a synthetic test dataset to test the fitting algorithms and to confirm the statistical outputs. We further establish a framework for the determination of multimodality throughout a protein system by rigorous evaluation of multimodal fits across all peptide spectra. We demonstrate this approach using entire protein datasets to detect multimodality, conformational heterogeneity, and characterize dynamics of small heat shock protein HSPB5 and two disease mutants.

A critical step in infections is the attachment of microorganisms to host cells using lectins that bind glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, an adhesin in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Distinct monoclonal antibodies that alter FimH glycan binding are available, but the mechanisms of action remain unclear. Here, we use cryo-electron microscopy, mass spectrometry, adhesion assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study demonstrates four mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for antimicrobials that target adhesins.

Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or αB-crystallin) is among the most widely expressed of the 10 human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at high levels. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between nonactivated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in nonactivated states. Either mutation or an activating pH change causes rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. Although beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased coaggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

Abstract BACKGROUND: BRCA1, a central player in gynecological cancers, collaborates with BARD1 to shape a potent complex pivotal for DNA binding and ubiquitin E3 ligase activities. This complex impacts a myriad of biological pathways. Notably, BRCA1-BARD1’s role in tumor suppression and homology-directed DNA repair (HDR) has been spotlighted. Our innovative approach used RING-domain mutations to craft ligase-dead BRCA1-BARD1 mutants, hypothesizing that these mutants would offer fresh insights into the DNA repair dynamics of BRCA1-BARD1. METHODS: Full length BRCA1-BARD1 or truncated mutants, and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. Stable mammalian cell lines HeLa and MDA-MB-436 that express wild type or mutant forms of BRCA1 and BARD1 were established for a host of analyses, including cellular fractionation, foci analysis, Proximity ligation assay (PLA), comet assays and clonogenic survival assays with various DNA damage agents. RESULTS: By systematic biochemical screening and a series of in vitro assays, we generated a truly BRCA1-BARD1 E3 dead mutant, BRCA1I26A, L63A, K65A-BARD1 (BRCA1-E3d), which lacks E3 ligase activity but possesses all other known attributes such as retention of BRCA1-BARD1 heterodimeric structure formation, DNA binding, and intact RAD51-mediated recombinase activity. To our surprise, we discovered that previously described BRCA1-BARD1 RING-domain mutant (BRCA1-I26A) still possessed ubiquitylation activity not found in BRCA1-E3d. To determine the biological significance of these mutants, cells stably expressing BRCA1-E3d were treated with various DNA-damaging agents and shown to more sensitive than WT or previously identified mutants. DNA repair pathway reporter assays determined these cells were deficient in various repair pathways compared to their BRCA1-WT counterparts. Further studies demonstrate that BRCA1-BARD1 E3 ligase is required for DNA resection during HDR, as evidenced in reduced levels of DNA repair-related foci formation such as RPA, RAD51 and CtIP in BRCA1-E3d cells. Furthermore, compared to BRCA1-WT cells, BRCA1-E3d cells were more sensitive to DNA damage reagents after depletion of 53BP1, which indicates BRCA1-BARD1 E3 ligase function also contributes to later stages of DNA repair completion. CONCLUSIONS: Our work dispels prevailing ambiguities surrounding BRCA1-BARD1 E3 ligase functions, underscoring its paramountcy in genome repair. This trailblazing research not only enriches our understanding but also beckons therapeutic interventions targeting tumor suppression. The unveiling of BRCA1-BARD1 E3 ligase's intricate regulatory dynamics combined with our novel mutants paves the way for an exciting new era in cancer therapeutics, hinting at superior treatments to enhance patient recovery. Citation Format: Wenjing Li, Meiling Wang, Nozomi Tomimatsu, Jae-Hoon Ji, Salvador Alejo, Gangadhara R. Sareddy, Sandeep Burma, Rachel Klevit, Weixing Zhao. Unraveling the Role of BRCA1-BARD1 E3 Ubiquitin Ligase in DNA Repair: A Promise for Enhanced Chemotherapy Outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-08.

Small Ubiquitin-related modifiers of the SUMO family regulate thousands of proteins in eukaryotic cells. Many SUMO substrates, effectors and enzymes carry short motifs (SIMs) that mediate low affinity interactions with SUMO proteins. This raises the question how specificity is achieved in target selection, SUMO paralogue choice and SUMO-dependent interactions. A unique but poorly understood feature of SUMO proteins is their intrinsically disordered N-terminus. We reveal a function for N-termini of human, C. elegans, and yeast SUMO proteins as intramolecular inhibitors of SUMO-SIM interactions. Mutational analyses, NMR spectroscopy, and Molecular Dynamics simulations indicate that SUMO’s N-terminus can inhibit SIM binding by fast and fuzzy interactions with SUMO‘s core. Deletion of the C. elegans SUMO1 N-terminus leads to p53-dependent apoptosis during germline development, indicating an important role of SUMO’s N-termini in DNA damage repair. Our findings reveal a mechanism of disorder-based autoinhibition that contributes to the specificity of SUMOylation and SUMO-dependent interactions.

Light-scattering protein aggregates in the eye lens cause cataract, one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover and has therefore evolved remarkable mechanisms to resist protein aggregation for a lifetime. Lens cytoplasm becomes progressively more oxidizing with age and especially with cataract onset. Proteomic and in vitro studies have converged on the importance of lens γ-crystallin misfolding via formation of non-native disulfide bonds for light scattering aggregation. We have now found that one of the evolved mechanisms of lens resilience involves an unusually abundant lens metabolite, myo-inositol, that suppresses oxidative γ-crystallin aggregation without being redox-active itself. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo­inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that myo-inositol is severely depleted in human cataractous lenses compared to age-matched controls, maintaining or restoring healthy levels of this compound in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1-nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Legends for Supplementary Figures S1-S13.

The tumor-suppressor proteins BRCA1 and BARD1 function as an E3 ubiquitin ligase to facilitate transcriptional repression and DNA damage repair. This is mediated in-part through its ability to mono-ubiquitylate histone H2A in nucleosomes. Studies in Caenorhabditis elegans have been used to elucidate numerous functions of BRCA1 and BARD1; however, it has not been established that the C. elegans orthologs, BRC-1 and BRD-1, retain all the functions of their human counterparts. Here we explore the conservation of enzymatic activity toward nucleosomes which leads to repression of estrogen-metabolizing cytochrome P450 (cyp) genes in humans. Biochemical assays establish that BRC-1 and BRD-1 contribute to ubiquitylation of histone H2A in the nucleosome. Mutational analysis shows that while BRC-1 likely binds the nucleosome using a conserved interface, BRD-1 and BARD1 have evolved different modes of binding, resulting in a difference in the placement of ubiquitin on H2A. Gene expression analysis reveals that in spite of this difference, BRC-1 and BRD-1 also contribute to cyp gene repression in C. elegans. Establishing conservation of these functions in C. elegans allows for use of this powerful model organism to address remaining questions regarding regulation of gene expression by BRCA1 and BARD1.

Background: Breast cancer type 1 susceptibility protein (BRCA1) is a tumor suppressor gene involved in DNA double strand break repair with well-known cancer implications. BRCA1 heterodimerizes with BRCA1 associated Ring domain 1 (BARD1) to form a complex with DNA binding and ubiquitin E3 ligase function capable of interacting with proteins of diverse biological processes, most notably homology-directed DNA repair. During DNA repair, BRCA1-BARD1 directly interfaces with nucleosomes and transfers mono ubiquitin (Ub) to lysine residues on the C-terminal tail of histone H2A. Although truncation of the enzymatic BRCA1-BARD1 RING-RING domain retains H2A ubiquitylating activity, full-length BRCA1-BARD1 binds more tightly with nucleosomes and displays higher H2A-Ub activity. However, the molecular basis and biological significance for this enhanced nucleosome binding and H2A-Ub activity is uncharacterized. Methods: Full length BRCA1-BARD1 or truncated mutants and histones were purified from E. coli. or insect cells. Nucleosomes were assembled for in vitro ubiquitylation reaction and binding assays. To determine the biological significance, mammalian cell lines that stably express wild type or mutant forms of BARD1 were established for cellular fractionation, foci analysis, and clonogenic survival studies alongside various DNA damage agents. Results: Our results show multiple interaction sites exist between BRCA1-BARD1 and nucleosomes which allow high-affinity chromatin binding and promote increased histone H2A ubiquitylation activity. Multivalent BARD1-nucleosome interactions, namely those using strong binding motifs located in the intrinsically disordered region (IDR) of BARD1, and the weak “kiss” interaction mediated by the RING domains of both BRCA1 and BARD1, are essential for H2A ubiquitylation by BRCA1-BARD1. Further, we isolated two types of specific histone binding and/or ubiquitylation-defective mutants of BARD1: a BARD1-IDR mutant with disrupted nucleosome binding withe retained H2A ubiquitylation ability, and a RING mutant that solely impairs H2A ubiquitylation. In both cases, we demonstrate that these mutants are hypersensitive to DNA damage agents, including polyADP-ribose polymerase (PARP) inhibitors, and demonstrate reduced capacity of BARD1 to associate with chromatin and foci formation owing to attenuated repair capacity. Conclusion: Our studies provide convincing evidence BRCA1-BARD1 interacts with nucleosomes and ubiquitylates histones via its E3 ligase activity. Further, it plays a critical role in DNA damage response and repair that contributes to genome stability, which when disrupted sensitizes them to DNA damage agents. Our results open new avenues towards understanding whether and how these mutations in BRCA1-BARD1 affect its tumor suppression functions and their implications clinically, ultimately with the goal to translate these findings for the benefit of cancer patients. Citation Format: Wenjing Li, Samuel R. Witus, Meiling Wang, Peter S. Brzovic, Rachel E. Klevit, Weixing Zhao. BRCA1-BARD1 ubiquitylates histones for genome maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6096.