scholarly journals Structural Insights into Mechanism of Antibiotic Regulation in Streptomyces

2014 ◽  
Vol 70 (a1) ◽  
pp. C700-C700
Author(s):  
Ruchi Anand ◽  
Hussain Bhukya
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Antibiotic Production ◽  
Promoter Sequence ◽  
Binding Mode ◽  
Biologically Active ◽  
Streptomyces Species ◽  
Structural Mechanism ◽  
A Genome

Streptomyces species are well-known for their wide variety of biologically active secondary metabolites and contribute to two-third of naturally occurring antibiotics. Production of antibiotics and resistance pathways in these species are dictated by interplay of transcriptional regulatory proteins that trigger downstream responses to either small diffusible molecules (autoinducers) or by binding to the antibiotic intermediates. These regulators have a ligand binding site and a DNA binding site and they carry out their transcription regulation via conformational changes induced upon ligand or DNA binding. To decipher the structural mechanism of action here we present the crystal structure of CprB in complex with its consensus DNA element to a resolution of 3.2 Å. The structure revealed that CprB belongs to the tetracycline family of antibiotic resistance efflux pumps regulators. CprB binds to the DNA as a tetramer via the helix-turn-helix (HTH) motif with the mode of DNA binding is most analogous to that observed for the broad spectrum multidrug resistance regulator QacR from Staphylococcus aureus. The binding of the DNA induces the restructuring of the CprB dimeric interface, thereby inducing a pendulum like motion of the HTH motif that inserts into the major grove of the DNA. A genome wide search for the cognate DNA element revealed that CprB serves as an autoregulatory protein and binds to its own promoter sequence. Our studies suggest that CprB is a part of a network of proteins that regulate the antibiotic production and resistance pathways in Streptomyces. Fluorescence anisotropy lifetime studies performed with both consensus and CprB promoter helped in concluding that both the sequence have an analogous mode of binding with the CprB DNA exhibiting a stronger binding profile as supported by ITC studies. A sequential binding mode, similar to a clamp and click model of binding was proposed.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules

2020 ◽  
Vol 6 (43) ◽  
pp. eabd7182
Author(s):  
Xingzhe Yao ◽  
Chao Chen ◽  
Yefei Wang ◽  
Sheng Dong ◽  
Ya-Jun Liu ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Protein Function ◽  
Titration Calorimetry ◽  
Resonance Spectroscopy ◽  
Biological Regulation ◽  
Structural Mechanism ◽  
Functional Sites ◽  
Ph Dependent

Many important proteins undergo pH-dependent conformational changes resulting in “on-off” switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from Clostridium acetobutylicum, which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4.8 and 7.5 with a 180° rotation of the bound dockerin. Combined analysis by nuclear magnetic resonance spectroscopy, crystal structure determination, mutagenesis, and isothermal titration calorimetry elucidates the chemical and structural mechanism of the pH-dependent switching of the binding sites. The pH-dependent dual-binding-site switch not only represents an elegant example of biological regulation but also provides a new approach for developing pH-dependent protein devices and biomaterials beyond an on-off switch for biotechnological applications.

scholarly journals Spectroscopic and electrochemical study of interactions between DNA and different salts of 1,4-dihydropyridine AV-153

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10061
Author(s):  
Elina Leonova ◽  
Karlis Shvirksts ◽  
Vitalijs Borisovs ◽  
Edgars Smelovs ◽  
Jelizaveta Sokolovska ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Biological Activities ◽  
Binding Mode ◽  
Water Soluble ◽  
Dna Bases ◽  
Spectroscopy Data ◽  
Vis Spectroscopy ◽  
Intercalation Binding ◽  
Phosphate Groups

1,4-dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antimutagenic and DNA-binding activity. The latter activity was first described for water-soluble 1,4-DHP with carboxylic group in position 4, the sodium salt of the 1,4-DHP derivative AV-153 among others. Some data show the modification of physicochemical properties and biological activities of organic compounds by metal ions that form the salts. We demonstrated the different affinity to DNA and DNA-protecting capacity of AV-153 salts, depending on the salt-forming ion (Na, K, Li, Rb, Ca, Mg). This study aimed to use different approaches to collate data on the DNA-binding mode of AV-153-Na and five other AV-153 salts. All the AV-153 salts in this study quenched the ethidium bromide and DNA complex fluorescence, which points to an intercalation binding mode. For some of them, the intercalation binding was confirmed using cyclic voltammetry and circular dichroism spectroscopy. It was shown that in vitro all AV-153 salts can interact with four DNA bases. The FTIR spectroscopy data showed the interaction of AV-153 salts with both DNA bases and phosphate groups. A preference for base interaction was observed as the AV-153 salts interacted mostly with G and C bases. However, the highest differences were detected in the spectral region assigned to phosphate groups, which might indicate either conformational changes of DNA molecule (B form to A or H form) or partial denaturation of the molecule. According to the UV/VIS spectroscopy data, the salts also interact with the human telomere repeat, both in guanine quadruplex (G4) and single-stranded form; Na and K salts manifested higher affinity to G4, Li and Rb –to single-stranded DNA.

scholarly journals If You Cannot Win Them, Join Them: Understanding New Ways to Target STAT3 by Small Molecules

2019 ◽  
Author(s):  
Francesc Sabanés ◽  
Joao Victor de Souza Cunha ◽  
Roger Estrada-Tejedor ◽  
Agnieszka K Bronowska
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Small Molecule ◽  
Poor Prognosis ◽  
Critical Role ◽  
Binding Mode ◽  
Pharmacological Intervention ◽  
Inhibition Mechanism ◽  
Structure Dynamics ◽  
Small Molecule Ligands

<p>Signal transducer activator of transcription 3 (STAT3) is among the most investigated oncogenic transcription factors, as it is highly associated with cancer initiation, progression, metastasis, chemoresistance, and immune evasion. The recent evidence from both preclinical and clinical studies have demonstrated that STAT3 plays a critical role in several malignancies associated with poor prognosis such as glioblastoma and triple-negative breast cancer (TNBC), and STAT3 inhibitors have shown efficacy in inhibiting cancer growth and metastasis. Constitutive activation of STAT3 by mutations occurs frequently in tumour cells, and directly contributes to many malignant phenotypes. Unfortunately, detailed structural biology studies on STAT3 as well as target-based drug discovery efforts have been hampered by difficulties in the expression and purification of the full length STAT3 and a lack of ligand-bound crystal structures.</p><p>Considering these, molecular modelling and simulations offer an attractive strategy for the assessment of “druggability” of STAT3 dimers and allow investigations of reported activating and inhibiting STAT3 mutants at the atomistic level of detail. In the present study, we focused on the effects exerted by reported STAT3 mutations on the protein structure, dynamics, DNA binding and dimerisation, thus linking structure, dynamics, energetics, and the biological function. By employing atomistic molecular dynamics (MD) and umbrella sampling (US) simulations to a series of human STAT3 dimer, which comprised wild-type protein and four mutations; we explained the modulation of STAT3 activity by these mutations. Counter-intuitively, our results show that D570K inhibitory mutation exerts its effect by enhancing rather than weakening STAT3-DNA interactions, which interferes with the DNA release by the protein dimer and thus inhibits STAT3 function as a transcription factor. We mapped the binding site and characterised the binding mode of a clinical candidate napabucasin/BBI-608 at STAT3, which resembles the effect of D570K mutation. We also discovered a novel binding site at STAT3 DNA binding domain, amenable to bind small molecule ligands.</p><p>Our results contribute to understanding the activation/inhibition mechanism of STAT3, to explain the molecular mechanism of STAT3 inhibition by napabucasin/BBI-608. In this study, alongside the characterisation of the BBI-608 binding mode, we mapped a novel binding sites amenable to bind small molecule ligands, which may pave the way to design novel STAT3 inhibitors and to suggest new strategies for pharmacological intervention to combat cancers associated with poor prognosis.</p>

scholarly journals Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter

2009 ◽  
Vol 418 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Marianne A. Grant ◽  
Rebecca M. Baron ◽  
Alvaro A. Macias ◽  
Matthew D. Layne ◽  
Mark A. Perrella ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
High Mobility ◽  
Promoter Sequence ◽  
Therapeutic Benefit ◽  
Mobility Shift ◽  
Beneficial Effects ◽  
The Core

The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with Gram-negative bacteria that elaborate endotoxin. The HMGA1 (high-mobility group A1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct (octamer) sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule MGB (minor-groove binder) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. We therefore hypothesized that netropsin would improve survival from murine endotoxaemia by attenuating NOS2 induction through interference with HMGA1 DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxaemia in wild-type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1 DNA-binding site was critical for this effect. EMSAs (electrophoretic mobility-shift assays) demonstrated that netropsin interferes with HMGA1 NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, NOESY data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1-binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modelling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes that are important for the development of critical illness.

scholarly journals Machine Learning to Identify Flexibility Signatures of Class A GPCR Inhibition

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 454 ◽  
Author(s):  
Joseph Bemister-Buffington ◽  
Alex J. Wolf ◽  
Sebastian Raschka ◽  
Leslie A. Kuhn
Keyword(s):  
Machine Learning ◽  
Ligand Binding ◽  
Binding Site ◽  
Nearest Neighbor ◽  
Three Dimensional ◽  
Binding Mode ◽  
Biologically Active ◽  
Ligand Binding Site ◽  
K Nearest Neighbor ◽  
Class A

We show that machine learning can pinpoint features distinguishing inactive from active states in proteins, in particular identifying key ligand binding site flexibility transitions in GPCRs that are triggered by biologically active ligands. Our analysis was performed on the helical segments and loops in 18 inactive and 9 active class A G protein-coupled receptors (GPCRs). These three-dimensional (3D) structures were determined in complex with ligands. However, considering the flexible versus rigid state identified by graph-theoretic ProFlex rigidity analysis for each helix and loop segment with the ligand removed, followed by feature selection and k-nearest neighbor classification, was sufficient to identify four segments surrounding the ligand binding site whose flexibility/rigidity accurately predicts whether a GPCR is in an active or inactive state. GPCRs bound to inhibitors were similar in their pattern of flexible versus rigid regions, whereas agonist-bound GPCRs were more flexible and diverse. This new ligand-proximal flexibility signature of GPCR activity was identified without knowledge of the ligand binding mode or previously defined switch regions, while being adjacent to the known transmission switch. Following this proof of concept, the ProFlex flexibility analysis coupled with pattern recognition and activity classification may be useful for predicting whether newly designed ligands behave as activators or inhibitors in protein families in general, based on the pattern of flexibility they induce in the protein.

scholarly journals Machine Learning to Identify Flexibility Signatures of Class A GPCR Inhibition

2020 ◽  
Author(s):  
Joseph Bemister-Buffington ◽  
Alex J. Wolf ◽  
Sebastian Raschka ◽  
Leslie A. Kuhn
Keyword(s):  
Machine Learning ◽  
Ligand Binding ◽  
Binding Site ◽  
Nearest Neighbor ◽  
Binding Mode ◽  
Biologically Active ◽  
Ligand Binding Site ◽  
K Nearest Neighbor ◽  
Graph Theoretic ◽  
Class A

AbstractWe show that machine learning can pinpoint features distinguishing inactive from active states in proteins, in particular identifying key ligand binding site flexibility transitions in GPCRs that are triggered by biologically active ligands. Our analysis was performed on the helical segments and loops in 18 inactive and 9 active class A GPCRs. These 3-dimensional structures were determined in complex with ligands. However, considering the flexible versus rigid state identified by graph-theoretic ProFlex rigidity analysis for each helix and loop segment with the ligand removed, followed by feature selection and k-nearest neighbor classification, was sufficient to identify four segments surrounding the ligand binding site whose flexibility/rigidity accurately predicts whether a GPCR is in an active or inactive state. GPCRs bound to inhibitors were similar in their pattern of flexible versus rigid regions, whereas agonist-bound GPCRs were more flexible and diverse. This new ligand-proximal flexibility signature of GPCR activity was identified without knowledge of the ligand binding mode or previously defined switch regions, while being adjacent to the known transmission switch. Following this proof of concept, the ProFlex flexibility analysis coupled with pattern recognition and activity classification may be useful for predicting whether newly designed ligands behave as activators or inhibitors, based on the pattern of flexibility they induce in the protein.

scholarly journals Genetic Analysis of Phage Mu Mor Protein Amino Acids Involved in DNA Minor Groove Binding and Conformational Changes

2011 ◽  
Vol 286 (41) ◽  
pp. 35852-35862 ◽  
Author(s):  
Muthiah Kumaraswami ◽  
Lakshmi Avanigadda ◽  
Rajendra Rai ◽  
Hee-Won Park ◽  
Martha M. Howe
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Binding Site ◽  
Conformational Changes ◽  
Minor Groove ◽  
Minor Groove Binding ◽  
Groove Binding ◽  
Specific Chemical ◽  
Mutant Proteins ◽  
Dna Minor Groove

Gene expression during lytic development of bacteriophage Mu occurs in three phases: early, middle, and late. Transcription from the middle promoter, Pm, requires the phage-encoded activator protein Mor and the bacterial RNA polymerase. The middle promoter has a −10 hexamer, but no −35 hexamer. Instead Pm has a hyphenated inverted repeat that serves as the Mor binding site overlapping the position of the missing −35 element. Mor binds to this site as a dimer and activates transcription by recruiting RNA polymerase. The crystal structure of the His-Mor dimer revealed three structural elements: an N-terminal dimerization domain, a C-terminal helix-turn-helix DNA-binding domain, and a β-strand linker between the two domains. We predicted that the highly conserved residues in and flanking the β-strand would be essential for the conformational flexibility and DNA minor groove binding by Mor. To test this hypothesis, we carried out single codon-specific mutagenesis with degenerate oligonucleotides. The amino acid substitutions were identified by DNA sequencing. The mutant proteins were characterized for their overexpression, solubility, DNA binding, and transcription activation. This analysis revealed that the Gly-Gly motif formed by Gly-65 and Gly-66 and the β-strand side chain of Tyr-70 are crucial for DNA binding by His-tagged Mor. Mutant proteins with substitutions at Gly-74 retained partial activity. Treatment with the minor groove- and GC-specific chemical chromomycin A3 demonstrated that chromomycin prevented His-Mor binding but could not disrupt a pre-formed His-Mor·DNA complex, consistent with the prediction that Mor interacts with the minor groove of the GC-rich spacer in the Mor binding site.

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