Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

Zannoni, Annamaria; Pelliciari, Simone; Musiani, Francesco; Chiappori, Federica; Roncarati, Davide; Scarlato, Vincenzo

doi:10.3390/ijms22157848

Cited by 8 publications

(16 citation statements)

References 51 publications

(84 reference statements)

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Section: Methodsmentioning

confidence: 99%

“…The previously obtained molecular models of dimeric HP1043 alone and bound to DNA were employed ( Zannoni et al, 2021 ). HP1043 model dimer bound to the DNA was obtained by a data-driven docking, employing NMR data, and domain X-ray deposited data, and the residues involved in DNA binding were experimentally validated (for details see ( Zannoni et al, 2021 )). Both complexes were relaxed through 100 ns of molecular dynamic simulation and the most representative conformations obtained from cluster analysis were employed for the virtual screening step.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant N-terminal His-tagged HP1043 wild-type and mutant proteins were overexpressed in Escherichia coli BL21 DE3 cells transformed with plasmid pTrc::1043 ( Supplementary Table S1 ). For electrophoretic mobility shift assays (EMSA), HP1043 was purified as previously described ( Zannoni et al, 2021 ). Briefly, the cells were incubated with lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, and 10 mM imidazole), lysozyme (0.5 mg/ml), and 1× cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland) for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Intriguingly, the study showed that HP1043 dimerizes and binds in vivo the promoter of genes involved in all the fundamental processes of the bacterial life cycle, namely, DNA replication, RNA transcription and translation, and energy production and conversion ( Pelliciari et al, 2017 ). The resulting head-to-head dimer conformation, different from the canonical head-to-tail of the OmpR/PhoB subfamily, was theoretically obtained, based on experimental mutagenesis data and inter-domain linker flexibility evaluation ( Zannoni et al, 2021 ). HP1043 appears to be fundamental for the fitness of the bacterium, a prerequisite for a successful infection, and it is a pivotal regulator on which H. pylori relies to modulate its metabolism and growing behavior.…”

Section: Introductionmentioning

confidence: 99%

“…Experimental data combined with the available HP1043 NMR structure were used as restraints to guide an in silico protein–DNA docking. The generated model shows an HP1043 dimer interacting in a head-to-head conformation with both the major and minor grooves of a target DNA sequence ( Zannoni et al, 2021 ). Moreover, the dimer conformation is known to increase the binding to the promoter region on DNA ( Simonovic and Volz, 2001 ).…”

Section: Introductionmentioning

confidence: 99%

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Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study

Antoniciello

Roncarati

Zannoni

et al. 2022

Front. Mol. Biosci.

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Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043–DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study

Antoniciello

Roncarati

Zannoni

et al. 2022

Front. Mol. Biosci.

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Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori

Åberg,

Gideonsson,

Bhat

et al. 2024

Nucleic Acids Research

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Adaptation to variations in pH is crucial for the ability of Helicobacter pylori to persist in the human stomach. The acid responsive two-component system ArsRS, constitutes the global regulon that responds to acidic conditions, but molecular details of how transcription is affected by the ArsR response regulator remains poorly understood. Using a combination of DNA-binding studies, in vitro transcription assays, and H. pylori mutants, we demonstrate that phosphorylated ArsR (ArsR-P) forms an active protein complex that binds DNA with high specificity in order to affect transcription. Our data showed that DNA topology is key for DNA binding. We found that AT-rich DNA sequences direct ArsR-P to specific sites and that DNA-bending proteins are important for the effect of ArsR-P on transcription regulation. The repression of sabA transcription is mediated by ArsR-P with the support of Hup and is affected by simple sequence repeats located upstream of the sabA promoter. Here stochastic events clearly contribute to the fine-tuning of pH-dependent gene regulation. Our results reveal important molecular aspects for how ArsR-P acts to repress transcription in response to acidic conditions. Such transcriptional control likely mediates shifts in bacterial positioning in the gastric mucus layer.

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2nd Workshop in Microbial Molecular Biology

Martı́nez

Brocchi

Balan

et al. 2023

Molecular Microbiology

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Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

Cited by 8 publications

References 51 publications

Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study

Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study

Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori

2nd Workshop in Microbial Molecular Biology

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