Structural basis of paralog-specific KDM2A/B nucleosome recognition.

The nucleosome acidic patch is a major interaction hub for chromatin, providing a platform for enzymes to dock and orient for nucleosome-targeted activities. To define the molecular basis of acidic patch recognition proteome wide, we performed an amino acid resolution acidic patch interactome screen. We discovered that the histone H3 lysine 36 (H3K36) demethylase KDM2A, but not its closely related paralog, KDM2B, requires the acidic patch for nucleosome binding. Despite fundamental roles in transcriptional repression in health and disease, the molecular mechanisms governing nucleosome substrate specificity of KDM2A/B, or any related JumonjiC (JmjC) domain lysine demethylase, remain unclear. We used a covalent conjugate between H3K36 and a demethylase inhibitor to solve cryogenic electron microscopy structures of KDM2A and KDM2B trapped in action on a nucleosome substrate. Our structures show that KDM2-nucleosome binding is paralog specific and facilitated by dynamic nucleosomal DNA unwrapping and histone charge shielding that mobilize the H3K36 sequence for demethylation.

Protein Kinase Signaling Networks Driven by Oncogenic Gq/11 in Uveal Melanoma Identified by Phosphoproteomic and Bioinformatic Analyses.

Metastatic uveal melanoma (UM) patients typically survive only 2 to 3 years because effective therapy does not yet exist. Here, to facilitate the discovery of therapeutic targets in UM, we have identified protein kinase signaling mechanisms elicited by the drivers in 90% of UM tumors: mutant constitutively active G protein α-subunits encoded by GNAQ (Gq) or GNA11 (G11). We used the highly specific Gq/11 inhibitor FR900359 (FR) to elucidate signaling networks that drive proliferation, metabolic reprogramming, and dedifferentiation of UM cells. We determined the effects of FR on the proteome and phosphoproteome of UM cells as indicated by bioinformatic analyses with CausalPath and site-specific gene set enrichment analysis. We found that inhibition of oncogenic Gq/11 caused deactivation of PKC, Erk, and the cyclin-dependent kinases CDK1 and CDK2 that drive proliferation. Inhibition of oncogenic Gq/11 in UM cells with low metastatic risk relieved inhibitory phosphorylation of polycomb-repressive complex subunits that regulate melanocytic redifferentiation. Site-specific gene set enrichment analysis, unsupervised analysis, and functional studies indicated that mTORC1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 drive metabolic reprogramming in UM cells. Together, these results identified protein kinase signaling networks driven by oncogenic Gq/11 that regulate critical aspects of UM cell biology and provide targets for therapeutic investigation.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling.

Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.

The Dark Kinase Knowledgebase: an online compendium of knowledge and experimental results of understudied kinases.

Kinases form the backbone of numerous cell signaling pathways, with their dysfunction similarly implicated in multiple pathologies. Further facilitated by their druggability, kinases are a major focus of therapeutic development efforts in diseases such as cancer, infectious disease and autoimmune disorders. While their importance is clear, the role or biological function of nearly one-third of kinases is largely unknown. Here, we describe a data resource, the Dark Kinase Knowledgebase (DKK; https://darkkinome.org), that is specifically focused on providing data and reagents for these understudied kinases to the broader research community. Supported through NIH's Illuminating the Druggable Genome (IDG) Program, the DKK is focused on data and knowledge generation for 162 poorly studied or 'dark' kinases. Types of data provided through the DKK include parallel reaction monitoring (PRM) peptides for quantitative proteomics, protein interactions, NanoBRET reagents, and kinase-specific compounds. Higher-level data is similarly being generated and consolidated such as tissue gene expression profiles and, longer-term, functional relationships derived through perturbation studies. Associated web tools that help investigators interrogate both internal and external data are also provided through the site. As an evolving resource, the DKK seeks to continually support and enhance knowledge on these potentially high-impact druggable targets.

Gain-of-function genetic screen of the kinome reveals BRSK2 as an inhibitor of the NRF2 transcription factor.

Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.

Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding.

Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition.

Enabling Hotspot Detection and Public Health Response to the COVID-19 Pandemic.

INTRODUCTION: Public-facing maps of COVID-19 cases, hospital admissions, and deaths are commonly displayed at the state, county, and zip code levels, and low case counts are suppressed to protect confidentiality. Public health authorities are tasked with case identification, contact tracing, and canvasing for educational purposes during a pandemic. Given limited resources, authorities would benefit from the ability to tailor their efforts to a particular neighborhood or congregate living facility. METHODS: We describe the methods of building a real-time visualization of patients with COVID-19-positive tests, which facilitates timely public health response to the pandemic. We developed an interactive street-level visualization that shows new cases developing over time and resolving after 14 days of infection. Our source data included patient demographics (ie, age, race and ethnicity, and sex), street address of residence, respiratory test results, and date of test. RESULTS: We used colored dots to represent infections. The resulting animation shows where new cases developed in the region and how patterns changed over the course of the pandemic. Users can enlarge specific areas of the map and see street-level detail on residential location of each case and can select from demographic overlays and contour mapping options to see high-level patterns and associations with demographics and chronic disease prevalence as they emerge. CONCLUSIONS: Before the development of this tool, local public health departments in our region did not have a means to map cases of disease to the street level and gain real-time insights into the underlying population where hotspots had developed. For privacy reasons, this tool is password-protected and not available to the public. We expect this tool to prove useful to public health departments as they navigate not only COVID-19 pandemic outcomes but also other public health threats, including chronic diseases and communicable disease outbreaks.

Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.

Positive Cooperativity in Substrate Binding by Human Thymidylate Synthase.

Thymidylate synthase (TS) catalyzes the production of the nucleotide dTMP from deoxyuridine monophosphate (dUMP), making the enzyme necessary for DNA replication and consequently a target for cancer therapeutics. TSs are homodimers with active sites separated by ∼30 Å. Reports of half-the-sites activity in TSs from multiple species demonstrate the presence of allosteric communication between the active sites of this enzyme. A simple explanation for the negative allosteric regulation occurring in half-the-sites activity would be that the two substrates bind with negative cooperativity. However, previous work on Escherichia coli TS revealed that dUMP substrate binds without cooperativity. To gain further insight into TS allosteric function, binding cooperativity in human TS is examined here. Isothermal titration calorimetry and two-dimensional lineshape analysis of NMR titration spectra are used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperativity between the two substrate binding events. We find that human TS binds dUMP with ∼9-fold entropically driven positive cooperativity (ρITC = 9 ± 1, ρNMR = 7 ± 1), in contrast to the apparent strong negative cooperativity reported previously. Our work further demonstrates the necessity of globally fitting isotherms collected under various conditions, as well as accurate determination of binding competent protein concentration, for calorimetric characterization of homotropic cooperative binding. Notably, an initial curvature of the isotherm is found to be indicative of positively cooperative binding. Two-dimensional lineshape analysis NMR is also found to be an informative tool for quantifying binding cooperativity, particularly in cases in which bound intermediates yield unique resonances.

Dasatinib Is Preferentially Active in the Activated B-Cell Subtype of Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and at least one-third of its patients relapse after treatment with the current chemotherapy regimen, R-CHOP. By gene-expression profiling, patients with DLBCL can be categorized into two clinically relevant subtypes: activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL. Patients with the ABC subtype have a worse prognosis than those with GCB, and the subtype is defined by chronic, over-active signaling through the B-cell receptor and NF-κB pathways. We examined the effects of the Src family kinase (SFK) inhibitor dasatinib in a panel of ABC and GCB DLBCL cell lines and found that the former are much more sensitive to dasatinib than the latter. However, using multiplexed inhibitor bead coupled to mass spectrometry (MIB/MS) kinome profiling and Western blot analysis, we found that both subtypes display inhibition of the SFKs in response to dasatinib after both short- and long-term treatment. The MIB/MS analyses revealed that several cell-cycle kinases, including CDK4, CDK6, and the Aurora kinases, are down-regulated by dasatinib treatment in the ABC, but not in the GCB, subtype. The present findings have potential implications for the clinical use of dasatinib for the treatment of ABC DLBCL, either alone or in combination with other agents.

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor.

Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

MSAcquisitionSimulator: data-dependent acquisition simulator for LC-MS shotgun proteomics.

UNLABELLED: Data-dependent acquisition (DDA) is the most common method used to control the acquisition process of shotgun proteomics experiments. While novel DDA approaches have been proposed, their evaluation is made difficult by the need of programmatic control of a mass spectrometer. An alternative is in silico analysis, for which suitable software has been unavailable. To meet this need, we have developed MSAcquisitionSimulator-a collection of C ++ programs for simulating ground truth LC-MS data and the subsequent application of custom DDA algorithms. It provides an opportunity for researchers to test, refine and evaluate novel DDA algorithms prior to implementation on a mass spectrometer. AVAILABILITY AND IMPLEMENTATION: The software is freely available from its Github repository http://www.github.com/DennisGoldfarb/MSAcquisitionSimulator/ which contains further documentation and usage instructions. CONTACT: weiwang@cs.ucla.edu or ben_major@med.unc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Spotlite: web application and augmented algorithms for predicting co-complexed proteins from affinity purification--mass spectrometry data.

Protein-protein interactions defined by affinity purification and mass spectrometry (APMS) suffer from high false discovery rates. Consequently, lists of potential interactions must be pruned of contaminants before network construction and interpretation, historically an expensive, time-intensive, and error-prone task. In recent years, numerous computational methods were developed to identify genuine interactions from the hundreds of candidates. Here, comparative analysis of three popular algorithms, HGSCore, CompPASS, and SAINT, revealed complementarity in their classification accuracies, which is supported by their divergent scoring strategies. We improved each algorithm by an average area under a receiver operating characteristics curve increase of 16% by integrating a variety of indirect data known to correlate with established protein-protein interactions, including mRNA coexpression, gene ontologies, domain-domain binding affinities, and homologous protein interactions. Each APMS scoring approach was incorporated into a separate logistic regression model along with the indirect features; the resulting three classifiers demonstrate improved performance on five diverse APMS data sets. To facilitate APMS data scoring within the scientific community, we created Spotlite, a user-friendly and fast web application. Within Spotlite, data can be scored with the augmented classifiers, annotated, and visualized ( http://cancer.unc.edu/majorlab/software.php ). The utility of the Spotlite platform to reveal physical, functional, and disease-relevant characteristics within APMS data is established through a focused analysis of the KEAP1 E3 ubiquitin ligase.

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models.

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. From a screen of human airway derived cell lines that express varying levels of ACE2/TMPRSS2, we found a subset that express comparably high endogenous levels of ACE2 but surprisingly did not support SARS-CoV-2 replication. Here we report that this resistance is mediated by a basally active cGAS-STING pathway culminating in interferon (IFN)-mediated restriction of SARS-CoV-2 replication at a post-entry step. Pharmacological inhibition of JAK1/2, depletion of the IFN-α receptor and cGAS-STING pathway effectors substantially increased SARS-CoV-2 replication in these cell models. While depletion of cGAS or STING was sufficient to reduce the preexisting levels of IFN-stimulated genes (ISGs), SARS-CoV-2 infection in STING knockout cells independently induced ISG expression. Remarkably, SARS-CoV-2-induced ISG expression in STING knockout cell as well as in primary human airway cultures was limited to uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway, but not viral sensing or IFN production, in productively infected cells. Of note, SARS-CoV-2-infected primary human airway cells also displayed markedly lower levels of STING expression, raising the possibility that SARS-CoV-2 can target STING expression or preferentially infect cells that express low levels of STING. Finally, ectopic ACE2 overexpression overcame the IFN-mediated blocks, suggesting the ability of SARS-CoV-2 to overcome these possibly saturable blocks to infection. Our study highlights that in addition to viral receptors, basal activation of the cGAS-STING pathway and innate immune defenses may contribute to defining SARS-CoV-2 cellular tropism.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wild-type (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract.

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.