Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

Unravelling the Interaction of Piperlongumine with the Nucleotide-Binding Domain of HSP70: A Spectroscopic and In Silico Study

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van’t Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M−1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.

Adenosine nucleotide binding sites on Complex V from diverse organisms

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

Closer vein spacing by ectopic expression of nucleotide-binding and leucine-rich repeat proteins in rice leaves

Key message Elevated expression of nucleotide-binding and leucine-rich repeat proteins led to closer vein spacing and higher vein density in rice leaves. Abstract To feed the growing global population and mitigate the negative effects of climate change, there is a need to improve the photosynthetic capacity and efficiency of major crops such as rice to enhance grain yield potential. Alterations in internal leaf morphology and cellular architecture are needed to underpin some of these improvements. One of the targets is to generate a “Kranz-like” anatomy in leaves that includes decreased interveinal spacing close to that in C4 plant species. As C4 photosynthesis has evolved from C3 photosynthesis independently in multiple lineages, the genes required to facilitate C4 may already be present in the rice genome. The Taiwan Rice Insertional Mutants (TRIM) population offers the advantage of gain-of-function phenotype trapping, which accelerates the identification of rice gene function. In the present study, we screened the TRIM population to determine the extent to which genetic plasticity can alter vein density (VD) in rice. Close vein spacing mutant 1 (CVS1), identified from a VD screening of approximately 17,000 TRIM lines, conferred heritable high leaf VD. Increased vein number in CVS1 was confirmed to be associated with activated expression of two nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. Overexpression of the two NB-LRR genes individually in rice recapitulates the high VD phenotype, due mainly to reduced interveinal mesophyll cell (M cell) number, length, bulliform cell size and thus interveinal distance. Our studies demonstrate that the trait of high VD in rice can be achieved by elevated expression of NB-LRR proteins limited to no yield penalty.

A Review on Nucleotide Binding Site – Leucine Rich Repeats Disease Resistance Genes in legumes

The crop plants of the family Leguminosae are second to cereal crops of commercial importance on the basis of area harvested and total production worldwide. It is well known globally that many crops do not give good yield due to certain diseases existing in their plants. Nowadays, there is much emphasis on developing disease resistant varieties of crops and especially of commercial crops. Plants need to protect themselves against attack from viruses, microbes, invertebrates and even other plants. NBS-LRR (Nucleotide binding site-leucine rich repeats) genes belong to the largest plant disease resistance gene family and are responsible for plant resistance to pathogens. Studies of the NBS-LRR gene family in plants represent an intriguing challenge and can provide knowledge on the genomic and molecular mechanisms that form the basis of gene regulation and protein function. Their evolution at the gene and genomic level can be defined through ancient and numerous gene families. In the present study, beneficial concepts for generating basic and fundamental knowledge on the NBS-LRR plant disease resistance genes are discussed with emphasis on selected legume plants of commercial importance.

The Complex Interplay between Autophagy and NLRP3 Inflammasome in Renal Diseases

Autophagy is a highly conserved process of the eukaryotic cell cycle. It plays an important role in the survival and maintenance of cells by degrading organelles, proteins, and macromolecules in the cytoplasm and the circulation of degraded products. The dysfunction of autophagy can lead to the pathology of many human diseases. The nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome belongs to the family of nucleotide-binding and oligomerization domain-like receptors (NLRs) and can induce caspase-1 activation, thus leading to the maturation and secretion of interleukin-1beta (IL-1β) and IL-18. It has been reported that the interplay between autophagy and NLRP3 inflammasome is involved in many diseases, including renal diseases. In this review, the interplay between autophagy and the NLRP3 inflammasome and the mechanisms in renal diseases are explored to provide ideas for relevant basic research in the future.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis. Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

Comparison of carbohydrate ABC importers from Mycobacterium tuberculosis

Background Mycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions. Results Through the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge. Conclusions The presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.