Browsing by Author "Cavanagh, John"
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Item Open Access A DNA mimic: the structure and mechanism of action for the anti-repressor protein AbbA.(Journal of molecular biology, 2014-05) Tucker, Ashley T; Bobay, Benjamin G; Banse, Allison V; Olson, Andrew L; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Varney, Kristen M; Losick, Richard; Cavanagh, JohnBacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function.Item Open Access Binding site on human immunoglobulin G for the affinity ligand HWRGWV.(Journal of molecular recognition : JMR, 2010-05) Yang, Haiou; Gurgel, Patrick V; Williams, D Keith; Bobay, Benjamin G; Cavanagh, John; Muddiman, David C; Carbonell, Ruben GAffinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383-Asn389 (SNGQPEN) located in the C(H)3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG.Item Open Access Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum.(Biomolecular NMR assignments, 2014-04) Bobay, Benjamin G; Thompson, Richele J; Milton, Debra L; Cavanagh, JohnVibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.Item Open Access Dynamics and activation in response regulators: the β4-α4 loop.(Biomolecular concepts, 2012-02) Bobay, Benjamin G; Hoch, James A; Cavanagh, JohnTwo-component signal transduction systems of microbes are a primary means to respond to signals emanating from environmental and metabolic fluctuations as well as to signals coordinating the cell cycle with macromolecular syntheses, among a large variety of other essential roles. Signals are recognized by a sensor domain of a histidine kinase which serves to convert signal binding to an active transmissible phosphoryl group through a signal-induced ATP-dependent autophosphorylation reaction directed to histidine residue. The sensor kinase is specifically mated to a response regulator, to which it transfers the phosphoryl group that activates the response regulator's function, most commonly gene repression or activation but also interaction with other regulatory proteins. Two-component systems have been genetically amplified to control a wide variety of cellular processes; for example, both Escherichia coli and Pseudomonas aeruginosa have 60 plus confirmed and putative two-component systems. Bacillus subtilis has 30 plus and Nostoc punctiformis over 100. As genetic amplification does not result in changes in the basic structural folds of the catalytic domains of the sensor kinase or response regulators, each sensor kinase must recognize its partner through subtle changes in residues at the interaction surface between the two proteins. Additionally, the response regulator must prepare itself for efficient activation by the phosphorylation event. In this short review, we discuss the contributions of the critical β4-α4 recognition loop in response regulators to their function. In particular, we focus on this region's microsecond-millisecond timescale dynamics propensities and discuss how these motions play a major role in response regulator recognition and activation.Item Open Access ¹H, ¹³C, and ¹⁵N resonance assignments and secondary structure prediction of the full-length transition state regulator AbrB from Bacillus anthracis.(Biomolecular NMR assignments, 2012-04) Olson, Andrew L; Bobay, Benjamin G; Melander, Christian; Cavanagh, JohnThe AbrB protein is a transcription factor that regulates the expression of numerous essential genes during the cells transition phase state. AbrB from Bacillus anthracis is, nototriously, the principal protein responsible for anthrax toxin gene expression and is highly homologous to the much-studied AbrB protein from Bacillus subtilis having 85% sequence identity and the ability to regulate the same target promoters. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the full-length AbrB protein from B. anthracis.Item Open Access Identification of BfmR, a response regulator involved in biofilm development, as a target for a 2-Aminoimidazole-based antibiofilm agent.(Biochemistry, 2012-12) Thompson, Richele J; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Peng, Lingling; Su, Zhaoming; Actis, Luis A; Melander, Christian; Cavanagh, John2-Aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI-BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.Item Open Access Long range dynamic effects of point-mutations trap a response regulator in an active conformation.(FEBS letters, 2010-10) Bobay, Benjamin G; Thompson, Richele J; Hoch, James A; Cavanagh, JohnWhen a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This 'conformational trapping' results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site.Item Open Access Structure and DNA-binding traits of the transition state regulator AbrB.(Structure (London, England : 1993), 2014-11) Olson, Andrew L; Tucker, Ashley T; Bobay, Benjamin G; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Cavanagh, JohnThe AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated a structure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding.Item Open Access Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds.(Molecular microbiology, 2017-10) Milton, Morgan E; Allen, C Leigh; Feldmann, Erik A; Bobay, Benjamin G; Jung, David K; Stephens, Matthew D; Melander, Roberta J; Theisen, Kelly E; Zeng, Daina; Thompson, Richele J; Melander, Christian; Cavanagh, JohnWith antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.Item Open Access The Solution Structures and Interaction of SinR and SinI: Elucidating the Mechanism of Action of the Master Regulator Switch for Biofilm Formation in Bacillus subtilis.(Journal of molecular biology, 2020-01) Milton, Morgan E; Draughn, G Logan; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Feldmann, Erik A; Thompson, Richele J; Myers, Katherine H; Santoro, Michael T; Kearns, Daniel B; Cavanagh, JohnBacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins, and DNA. The gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining high-resolution solution nuclear magnetic resonance (NMR), chemical cross-linking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased repressor activity. Finally, we provide an evidence-based mechanism that confirms how disruption of the SinR tetramer by SinI regulates gene expression.Item Open Access The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism.(Journal of molecular biology, 2018-03) Draughn, G Logan; Milton, Morgan E; Feldmann, Erik A; Bobay, Benjamin G; Roth, Braden M; Olson, Andrew L; Thompson, Richele J; Actis, Luis A; Davies, Christopher; Cavanagh, JohnThe rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed. Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.