FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Recognition of the Diglycine C-End Degron by CRL2KLHDC2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.

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

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

Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB.

Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding sites have been elucidated in Streptomyces venezuelae (Sven), where they coordinate to activate sporulation septation. However, how these factors cooperate at the molecular level is not understood. Here we present cryoelectron microscopy structures of Sven transcriptional regulatory complexes comprising RNA polymerase (RNAP) σA-holoenzyme and WhiA and WhiB, in complex with the WhiA/B target promoter sepX. These structures reveal that WhiB binds to domain 4 of σA (σA4) of the σA-holoenzyme, bridging an interaction with WhiA while making non-specific contacts with the DNA upstream of the -35 core promoter element. The N-terminal homing endonuclease-like domain of WhiA interacts with WhiB, while the WhiA C-terminal domain (WhiA-CTD) makes base-specific contacts with the conserved WhiA GACAC motif. Notably, the structure of the WhiA-CTD and its interactions with the WhiA motif are strikingly similar to those observed between σA4 housekeeping σ-factors and the -35 promoter element, suggesting an evolutionary relationship. Structure-guided mutagenesis designed to disrupt these protein-DNA interactions reduces or abolishes developmental cell division in Sven, confirming their significance. Finally, we compare the architecture of the WhiA/B σA-holoenzyme promoter complex with the unrelated but model CAP Class I and Class II complexes, showing that WhiA/WhiB represent a new mechanism in bacterial transcriptional activation.

Effects of charge on protein ion structure: Lessons from cation-to-anion, proton-transfer reactions.

Collision cross-section values, which can be determined using ion mobility experiments, are sensitive to the structures of protein ions and useful for applications to structural biology and biophysics. Protein ions with different charge states can exhibit very different collision cross-section values, but a comprehensive understanding of this relationship remains elusive. Here, we review cation-to-anion, proton-transfer reactions (CAPTR), a method for generating a series of charge-reduced protein cations by reacting quadrupole-selected cations with even-electron monoanions. The resulting CAPTR products are analyzed using a combination of ion mobility, mass spectrometry, and collisional activation. We compare CAPTR to other charge-manipulation strategies and review the results of various CAPTR-based experiments, exploring their contribution to a deeper understanding of the relationship between protein ion structure and charge state.

High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid.

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.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 µM) and low (Kd,2 > 10 µM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 µM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Metastability of Protein Solution Structures in the Absence of a Solvent: Rugged Energy Landscape and Glass-like Behavior.

Native ion mobility/mass spectrometry is well-poised to structurally screen proteomes but characterizes protein structures in the absence of a solvent. This raises long-standing unanswered questions about the biological significance of protein structures identified through ion mobility/mass spectrometry. Using newly developed computational and experimental ion mobility/ion mobility/mass spectrometry methods, we investigate the unfolding of the protein ubiquitin in a solvent-free environment. Our data suggest that the folded, solvent-free ubiquitin observed by ion mobility/mass spectrometry exists in a largely native fold with an intact ß-grasp motif and α-helix. The ensemble of folded, solvent-free ubiquitin ions can be partitioned into kinetically stable subpopulations that appear to correspond to the structural heterogeneity of ubiquitin in solution. Time-resolved ion mobility/ion mobility/mass spectrometry measurements show that folded, solvent-free ubiquitin exhibits a strongly stretched-exponential time dependence, which simulations trace to a rugged energy landscape with kinetic traps. Unfolding rate constants are estimated to be approximately 800 to 20,000 times smaller than in the presence of water, effectively quenching the unfolding process on the time scale of typical ion mobility/mass spectrometry measurements. Our proposed unfolding pathway of solvent-free ubiquitin shares substantial characteristics with that established for the presence of solvent, including a polarized transition state with significant native content in the N-terminal ß-hairpin and α-helix. Our experimental and computational data suggest that (1) the energy landscape governing the motions of folded, solvent-free proteins is rugged in analogy to that of glassy systems; (2) large-scale protein motions may at least partially be determined by the amino acid sequence of a polypeptide chain; and (3) solvent facilitates, rather than controls, protein motions.

Quantitatively Differentiating Antibodies Using Charge-State Manipulation, Collisional Activation, and Ion Mobility-Mass Spectrometry.

Antibody-based therapeutics continue to expand both in the number of products and in their use in patients. These heterogeneous proteins challenge traditional drug characterization strategies, but ion mobility (IM) and mass spectrometry (MS) approaches have eased the challenge of higher-order structural characterization. Energy-dependent IM-MS, e.g., collision-induced unfolding (CIU), has been demonstrated to be sensitive to subtle differences in structure. In this study, we combine a charge-reduction method, cation-to-anion proton-transfer reactions (CAPTR), with energy-dependent IM-MS and varied solution conditions to probe their combined effects on the gas-phase structures of IgG1κ and IgG4κ from human myeloma. CAPTR paired with MS-only analysis improves the confidence of charge-state assignments and the resolution of the interfering protein species. Collision cross-section distributions were determined for each of the charge-reduced products. Similarity scoring was used to quantitatively compare distributions determined from matched experiments analyzing samples of the two antibodies. Relative to workflows using energy-dependent IM-MS without charge-state manipulation, combining CAPTR and energy-dependent IM-MS enhanced the differentiation of these antibodies. Combined, these results indicate that CAPTR can benefit many aspects of antibody characterization and differentiation.

Global Effects of the Developmental Regulator BldB in Streptomyces venezuelae.

In Streptomyces, the Bld (Bald) regulators control formation of the reproductive aerial hyphae. The functions of some of these regulators have been well characterized, but BldB has remained enigmatic. In addition to the bldB gene itself, Streptomyces venezuelae has 10 paralogs of bldB that sit next to paralogs of whiJ and abaA. Transcriptome sequencing (RNA-seq) revealed that loss of BldB function causes the dramatic transcriptional upregulation of the abaA paralogs and a novel inhibitor of sporulation, iosA, and that cooverexpression of just two of these genes, iosA and abaA6, was sufficient to recapitulate the bldB mutant phenotype. Further RNA-seq analysis showed that the transcription factor WhiJ9 is required for the activation of iosA seen in the bldB mutant, and biochemical studies showed that WhiJ9 mediates the activation of iosA expression by binding to direct repeats in the iosA-whiJ9 intergenic region. BldB and BldB9 hetero-oligomerize, providing a potential link between BldB and the iosA-whiJ9-bldB9 locus. This work greatly expands our overall understanding of the global effects of the BldB developmental regulator. IMPORTANCE To reproduce and disperse, the filamentous bacterium Streptomyces develops specialized reproductive structures called aerial hyphae. The formation of these structures is controlled by the bld (bald) genes, many of which encode transcription factors whose functions have been characterized. An exception is BldB, a protein whose biochemical function is unknown. In this study, we gain insight into the global effects of BldB function by examining the genome-wide transcriptional effects of deleting bldB. We identify a small set of genes that are dramatically upregulated in the absence of BldB. We show that their overexpression causes the bldB phenotype and characterize a transcription factor that mediates the upregulation of one of these target genes. Our results provide new insight into how BldB influences Streptomyces development.

Are the Gas-Phase Structures of Molecular Elephants Enduring or Ephemeral? Results from Time-Dependent, Tandem Ion Mobility.

The structural stability of biomolecules in the gas phase remains an important topic in mass spectrometry applications for structural biology. Here, we evaluate the kinetic stability of native-like protein ions using time-dependent, tandem ion mobility (IM). In these tandem IM experiments, ions of interest are mobility-selected after a first dimension of IM and trapped for up to ∼14 s. Time-dependent, collision cross section distributions are then determined from separations in a second dimension of IM. In these experiments, monomeric protein ions exhibited structural changes specific to both protein and charge state, whereas large protein complexes did not undergo resolvable structural changes on the timescales of these experiments. We also performed energy-dependent experiments, i.e., collision-induced unfolding, as a comparison for time-dependent experiments to understand the extent of unfolding. Collision cross section values observed in energy-dependent experiments using high collision energies were significantly larger than those observed in time-dependent experiments, indicating that the structures observed in time-dependent experiments remain kinetically trapped and retain some memory of their solution-phase structure. Although structural evolution should be considered for highly charged, monomeric protein ions, these experiments demonstrate that higher-mass protein ions can have remarkable kinetic stability in the gas phase.

Perioperative factors influencing hospitalization duration for pediatric neck abscesses.

PURPOSE: Pediatric neck abscesses are a common pathology seen in an ambulatory setting. Although some pediatric neck abscesses are managed medically with antibiotics, surgical intervention is often required. Given the often non-emergent presentation of many abscesses, a variety of logistical and perioperative factors may delay time to care and subsequently prolong hospital stay. The objective of this study was to examine factors that influence the overall time to surgery (TTS) and hospital length of stay (LOS) in a pediatric population with neck abscesses who ultimately require surgical drainage. MATERIALS AND METHODS: 161 pediatric patients who underwent incision and drainage of a neck abscess over a ten-year period at a tertiary referral children's center were reviewed. Demographic information, radiographic studies, and surgical information were extracted from patient charts. Descriptive statistics, Mann-Whitney U tests, and multivariate analyses were performed. RESULTS: The most common subcategory location was deep neck abscesses (33.1 %). Computed tomography (CT) was the most common pre-operative imaging modality (54.1 %) followed by ultrasound (US) (49.1 %) and magnetic resonance imaging (2.6 %). US and a combination of multiple preoperative imaging modalities were associated with increased LOS and TTS. Repeat surgery was associated with increased LOS. Pre-admission antibiotic use was associated with increased LOS and TTS. Younger patients were more likely to have a longer LOS. CONCLUSIONS: A variety of factors can influence TTS, LOS, and time from surgery to discharge including patient age, abscess location, a non-optimized utilization of imaging modalities, the utilization of pre-admission antibiotics, and the need for repeat operations.

Genome-Wide Identification of the LexA-Mediated DNA Damage Response in Streptomyces venezuelae.

DNA damage triggers a widely conserved stress response in bacteria called the SOS response, which involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs, but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA-damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA-alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using surface plasmon resonance, we further confirmed the LexA DNA binding motif (SOS box) and demonstrated that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCE The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic-producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

Evaluating Equity Through the Social Determinants of Hearing Health.

Hearing loss in adults is a significant public health problem throughout the world. Undiagnosed and untreated hearing loss causes a measurable impact on health and social, occupational, and emotional well-being of those affected. In spite of a wide array of health care resources to identify and manage hearing loss, there exist vast disparities in outcomes, as well as access to and utilization of hearing healthcare. Hearing rehabilitation outcomes may vary widely among different populations and there is a pressing need to understand, in a broader sense, the factors that influence equitable outcomes, access, and utilization. These factors can be categorized according to the widely accepted framework of social determinants of health, which is defined by the World Health Organization as "the conditions in which people are born, grow, work, live, and age." According to Healthy People 2030, these determinants can be broken into the following domains: healthcare access and quality, education access and quality, social and community context, economic stability, and neighborhood and built environment. This article defines these domains and examines the published research and the gaps in research of each of these domains, as it pertains to hearing health and healthcare. Herein, we review foundational sources on the social determinants of health and hearing-related research focused on the topic. Further consideration is given to how these factors can be evaluated in a systematic fashion and be incorporated into translational research and hearing health care.

The Use of Person-centered Language in Medical Research Articles Focusing on Hearing Loss or Deafness.

BACKGROUND: Hearing loss represents one of the most common disabilities worldwide. Despite its prevalence, there is a degree of stigmatization within the public's perception of, or attitude toward, individuals diagnosed with hearing loss or deafness. This stigmatization is propagated by the way hearing loss is referenced, especially in writing. Although the medical community is familiar with hearing loss, medical research is not consistently compliant with nonstigmatizing terminology, like person-centered language (PCL). This study aims to quantify the use of PCL in medical research related to hearing loss. METHODS: A cross-sectional analysis of articles related to hearing loss was performed using PubMed as the primary search engine. The search encompassed articles from January 1, 2016, to November 17, 2020. Journals had to have at least 20 search returns to be included in this study. The primary search resulted in 2392 articles from 31 journals. The sample was then randomized and the first 500 articles were chosen for data extraction. Article screening was performed systematically. Each article was evaluated for predetermined non-PCL terminology to determine adherence to the American Medical Association Manual of Style (AMAMS) guidelines. Articles were included if they involved research with human participants and were available in English. Commentaries and editorials were excluded. RESULTS: Four hundred eighty-two articles were included in this study. Results from this study indicate that 326 articles were not adherent to AMAMS guidelines for PCL (326/482; 68%). Emotional language (i.e., burden, suffer, afflicted) was employed to reference hearing loss in 114 articles (114/482; 24%). Non-PCL adherent labels (i.e., impaired and handicapped) were identified in 46% (221/482) of articles related to hearing loss or deafness. Sixty-seven articles (67/482; 14%) used person-first language in reference to the word "deaf" and 15 articles (15/482; 3%) used "deaf" as a label. CONCLUSIONS: Based on the findings from this cross-sectional analysis, the majority of medical research articles that address hearing loss contain terminology that does not conform to PCL guidelines, as established by AMAMS. Many respected organizations, like the American Medical Association, have encouraged the use of PCL in interactions between patient and medical provider. This encompasses communication in person and in writing. This recommendation stems from the understood role that language plays in how we build impressions of others, especially in a medical context. Implementing PCL to destigmatize language used in reference to deafness or hearing loss is essential to increase advocacy and protect the autonomy of these individuals.

Expanding Clinical Trials Designs to Extend Equitable Hearing Care.

Clinical trials are critically important to translate scientific innovations into clinical practice. Hearing healthcare depends on this translational approach to improve outcomes and quality of life. Across the spectrum of healthcare, there is a lack of diverse participation in clinical trials, a failure to recruit and retain underrepresented and underserved populations, and an absence of rigorous dissemination and implementation of novel research to broader populations. The field of hearing healthcare research would benefit from expanding the types and designs of clinical trials that extend hearing healthcare and novel interventions to diverse populations, as well as emphasizing trials that evaluate factors influencing how that care can be delivered effectively. This article explores the following: (1) the role, value, and design types of clinical trials (randomized controlled, cluster randomized, stepped wedge, and mixed methods) to address health equity; (2) the importance of integrating community and stakeholder involvement; and (3) dissemination and implementation frameworks and designs for clinical trials (hybrid trial designs). By adopting a broader range of clinical trial designs, hearing healthcare researchers may be able to extend scientific discoveries to a more diverse population.

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster.

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd ) for the SvWhiD-σHrdB4 complex as ∼0.7 µm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8-10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

Defining the regulon of genes controlled by σE , a key regulator of the cell envelope stress response in Streptomyces coelicolor.

The extracytoplasmic function (ECF) σ factor, σE , is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation-sequencing (ChIP-seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE -binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin-binding proteins, two L,D-transpeptidases, a LytR-CpsA-Psr-family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE -mediated cell envelope stress response in the genus Streptomyces.

High-Precision, Gas-Phase Hydrogen/Deuterium-Exchange Kinetics by Mass Spectrometry Enabled by Exchange Standards.

Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using cosampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.