Insights into the Allosteric Response to Acidity by the Helicobacter pylori NikR Transcription Factor.

Helicobacter pylori NikR (HpNikR) is a nickel-responsive transcription factor that regulates genes involved in nickel homeostasis, which is essential for the survival of this pathogen within the acidic human stomach. HpNikR also responds to drops in pH and regulates genes controlling acid acclimation of the bacteria, independently of nickel. We previously showed that nickel binding biases the conformational ensemble of HpNikR to the more DNA-binding competent states via an allosteric network of residues encompassing the nickel binding sites and the interface between the metal- and DNA-binding domains. Here, we examine how acidity promotes this response using 19F-NMR, mutagenesis, and DNA-binding studies. 19F-NMR revealed that a drop in pH from 7.6 to 6.0 does little to shift the conformational ensemble of HpNikR to the DNA binding-compatible cis conformer. Nevertheless, DNA-binding affinities of apo-HpNikR at pH 6.0 and Ni(II)-HpNikR at pH 7.6 are comparable for the ureA promoter. Histidine residues of the nickel binding sites were shown to be important for pH-dependent DNA binding and thus likely impart positive charge to the protein, initiating long-range electrostatic interactions with DNA that induce DNA complexation. The results point to a different DNA-binding mechanism in response to acidity compared to the conformational selection mechanism in response to nickel and overall provide new insights into the influence of pH on HpNikR activity, which contributes to H. pylori viability.

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor.

In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H. pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states. Nickel binding slows down conformational exchange between these states and shifts the equilibrium toward the binding-competent states. The protein then becomes stabilized in a cis conformation upon binding the ureA promoter. Here, we investigate how nickel binding creates this response and how it is transmitted to the DNA-binding domains. Through mutagenesis, DNA-binding studies, and computational methods, the allosteric response to nickel was found to be propagated from the nickel-binding sites to the DNA-binding domains via the ß-sheets of the metal-binding domain and a network of residues at the inter-domain interface. Our computational results suggest that nickel binding increases protein rigidity to slow down the conformational exchange. A thymine base in the ureA promoter sequence, known to be critical for high affinity DNA binding by HpNikR, was also found to be important for the allosteric response, while a modified version of this promoter further highlighted the importance of the DNA sequence in modulating the response. Collectively, our results provide insights into regulation of a key protein for H. pylori survival.

Allosteric Mechanisms of Nonadditive Substituent Contributions to Protein-Ligand Binding.

Quantifying chemical substituent contributions to ligand-binding free energies is challenging due to nonadditive effects. Protein allostery is a frequent cause of nonadditivity, but the underlying allosteric mechanisms often remain elusive. Here, we propose a general NMR-based approach to elucidate such mechanisms and we apply it to the HCN4 ion channel, whose cAMP-binding domain is an archetypal conformational switch. Using NMR, we show that nonadditivity arises not only from concerted conformational transitions, but also from conformer-specific effects, such as steric frustration. Our results explain how affinity-reducing functional groups may lead to affinity gains if combined. Surprisingly, our approach also reveals that nonadditivity depends markedly on the receptor conformation. It is negligible for the inhibited state but highly significant for the active state, opening new opportunities to tune potency and agonism of allosteric effectors.

The impact of processing on the cytotoxicity of graphene oxide.

In-house prepared graphene oxide (GO) was processed via base washing, sonication, cleaning and combinations of these processing techniques to evaluate the impact on the flake morphology, composition and cytotoxicity of the material. The flakes of unprocessed GO were relatively planar, but upon base washing, the flakes became textured exhibiting many folds and creases observed by AFM. In addition to the pronounced effect on the topography, base washing increased the C/O ratio and increased the cytotoxicity of GO on all four cell lines studied determined via the WST-8 assay. Sonicating the unprocessed and base washed samples resulted in smaller flakes with a similar topography; the base washed flakes lost the texture previously observed upon sonication. The sonicated samples were more toxic than the unprocessed sample, attributed to the smaller flake size, but were interestingly less toxic than the base washed, unsonicated sample despite the base washed unsonicated sample having a larger flake size. This unexpected finding was confirmed by a second analyst using the same, and a different source of GO and resulted in the conclusion that the morphology of GO greatly impacts the cytotoxicity. Cleaning the GO reduced the amount of nitrogen and sulfur impurities in the sample but had no significant impact on the cytotoxicity of the material. It was observed that nutrient depletion via nanomaterial adsorption was not the route of cytotoxicity for the GO samples studied.

Assessing size-dependent cytotoxicity of boron nitride nanotubes using a novel cardiomyocyte AFM assay.

As boron nitride nanotubes (BNNTs) find increased use in numerous applications, potential adverse health effects of BNNT exposure are a growing concern. Current in vitro cytotoxicity studies on BNNTs are inconsistent and even contradictory, likely due to the lack of reference materials, standardized characterization methods and measurement protocols. New approaches, particularly with the potential to reliably relate in vitro to in vivo studies, are critically needed. This work introduces a novel atomic force microscopy (AFM)-based cardiomyocyte assay that reliably assesses the cytotoxicity of a well-characterized boron nitride nanotube reference material, code named BNNT-1. High energy probe sonication was used to modify and control the length of BNNT-1. The polymer polyethylenimine (PEI) was used concurrently with sonication to produce stable, aqueous dispersions of BNNT-1. These dispersions were used to perform a systematic analysis on both the length and height of BNNT-1 via a correlated characterization approach of dynamic light scattering (DLS) and AFM. Cytotoxicity studies using the novel cardiomyocyte AFM model were in agreement with traditional colorimetric cell metabolic assays, both revealing a correlation between tube length and cytotoxicity with longer tubes having higher cytotoxicity. In addition to the size-dependent cytotoxicity, it was found that BNNT-1 exhibits concentration and cell-line dependent cytotoxic effects.