Conserved Apical Proline Regulating the Structure and DNA Binding Properties of Helicobacter pylori Histone-like DNA Binding Protein (Hup).

Prokaryotic cells lack a proper dedicated nuclear arrangement machinery. A set of proteins known as nucleoid associated proteins (NAPs) perform opening and closure of nucleic acids, behest cellular requirement. Among these, a special class of proteins analogous to eukaryotic histones popularly known as histone-like (HU) DNA binding proteins facilitate the nucleic acid folding/compaction thereby regulating gene architecture and gene regulation. DNA compaction and DNA protection in Helicobacter pylori is performed by HU protein (Hup). To dissect and galvanize the role of proline residue in the binding of Hup with DNA, the structure-dynamics-functional relationship of Hup-P64A variant was analyzed. NMR and biophysical studies evidenced that Hup-P64A protein attenuated DNA-binding and induced structural/stability changes in the DNA binding domain (DBD). Moreover, molecular dynamics simulations and 15N relaxation studies established the reduced conformational dynamics of P64A protein. This comprehensive study dissected the exclusive role of evolutionarily conserved apical proline residue in regulating the structure and DNA binding of Hup protein as P64 is presumed to be involved in the external leverage mechanism responsible for DNA bending and packaging, as proline rings wedge into the DNA backbone through intercalation besides their significant role in DNA binding.

Molecular Insights into Conformational Heterogeneity and Enhanced Structural Integrity of Helicobacter pylori DNA Binding Protein Hup at Low pH.

The summarized amalgam of internal relaxation modulations and external forces like pH, temperature, and solvent conditions determine the protein structure, stability, and function. In a free-energy landscape, although conformers are arranged in vertical hierarchy, there exist several adjacent parallel sets with conformers occupying equivalent energy cleft. Such conformational states are pre-requisites for the functioning of proteins that have oscillating environmental conditions. As these conformational changes have utterly small re-arrangements, nuclear magnetic resonance (NMR) spectroscopy is unique in elucidating the structure-dynamics-stability-function relationships for such conformations. Helicobacter pylori survives and causes gastric cancer at extremely low pH also. However, least is known as to how the genome of the pathogen is protected from reactive oxygen species (ROS) scavenging in the gut at low pH under acidic stress. In the current study, biophysical characteristics of H. pylori DNA binding protein (Hup) have been elucidated at pH 2 using a combination of circular dichroism, fluorescence, NMR spectroscopy, and molecular dynamics simulations. Interestingly, the protein was found to have conserved structural features, differential backbone dynamics, enhanced stability, and DNA binding ability at low pH as well. In summary, the study suggests the partaking of Hup protein even at low pH in DNA protection for maintaining the genome integrity.

Epigallocatechin Gallate with Potent Anti-Helicobacter pylori Activity Binds Efficiently to Its Histone-like DNA Binding Protein.

Helicobacter pylori (H. pylori)-a human gastric pathogen-forms a major risk factor for the development of various gastric pathologies such as chronic inflammatory gastritis, peptic ulcer, lymphomas of mucosa-associated lymphoid tissues, and gastric carcinoma. The complete eradication of infection is the primary objective of treating any H. pylori-associated gastric condition. However, declining eradication efficiencies, off-target effects, and patient noncompliance to prolong and broad-spectrum antibiotic treatments has spurred the clinical interest to search for alternative effective and safer therapeutic options. As natural compounds are safe and privileged with high levels of antibacterial-activity, previous studies have tested and reported a plethora of such compounds with potential in vitro/in vivo anti-H. pylori activity. However, the mode of action of majority of these natural compounds is unclear. The present study has been envisaged to compile the information of various such natural compounds and to evaluate their binding with histone-like DNA-binding proteins of H. pylori (referred here as Hup) using in silico molecular docking-based virtual screening experiments. Hup-being a major nucleoid-associated protein expressed by H. pylori-plays a strategic role in its survival and persistent colonization under hostile stress conditions. The ligand with highest binding energy with Hup-that is, epigallocatechin-(-)gallate (EGCG)-was rationally selected for further computational and experimental testing. The best docking poses of EGCG with Hup were first evaluated for their solution stability using long run molecular dynamics simulations and then using fluorescence and nuclear magnetic resonance titration experiments which demonstrated that the binding of EGCG with Hup is fairly strong (the resultant apparent dissociation constant (k D) values were equal to 2.61 and 3.29 ± 0.42 µM, respectively).

Exquisite binding interaction of 18ß-Glycyrrhetinic acid with histone like DNA binding protein of Helicobacter pylori: A computational and experimental study.

The Histone-like DNA binding protein is one of the most abundant nucleoid associated protein expressed by human gastric-pathogen, Helicobacter pylori (H. pylori). The protein -referred here as Hup- has been recognized as a potential drug target for developing therapeutic strategies against H. pylori. However, no attempts have been made, so far, to perturb the functioning of Hup through small molecules. As a first step in this direction, we virtually screened a natural product library containing 56 drug-like bioactive compounds and rationally selected 18ß-Glycyrrhetinic acid (GrA) for further computational and experimental testing of its binding interaction with Hup at the molecular level. The binding modes for GrA-Hup complexes were identified using in silico molecular docking methods and their solution dynamics and stability were evaluated using long run molecular dynamics simulations. Next, we experimentally demonstrated this binding interaction using fluorescence-quenching and ligand based NMR approaches. The fluorescence quenching and NMR titration experiments resulted into apparent dissociation constant (kD) for GrA-Hup binding equal to 87±12 µM and 36.6±1.5 µM, respectively. The various results demonstrate that GrA exhibits an exquisite binding interaction with Hup and would serve as an important molecular scaffold for developing next generation anti-H. pylori agents.

Effect of urea concentration on instant refolding of Nuclear Export Protein (NEP) from Influenza-A virus H1N1: A solution NMR based investigation.

Nuclear-export-protein (NEP) plays multiple-functions during influenza virus replication-cycle and shows unique pattern of conserved residues, which altogether make NEP a potential target for developing novel anti-influenza drugs. However, the mechanistic structural biology of NEP has not been fully characterized so far owing to its tendency to aggregate in solution. As structural information is important to guide rational drug-discovery process; therefore, procedural optimization efforts are going on to achieve properly folded NEP in sub-millimolar concentrations for solution-NMR investigations. As a first step in this direction, the refolding-cum-aggregation behavior of recombinant-NEP with N-terminal purification-tag (referred here as NEPN) at different urea-concentrations has been investigated here by NMR-based methods. Several attempts were made to refold denatured NEP-N through step-dialysis. However, owing to its strong tendency to aggregate, excessive precipitation was observed at sub-higher levels of urea concentration (5.0 ± 1.0 M). Finally, we used drip-dilution method with 10.5 M urea-denatured NEP-N and were able to refold NEP-N instantly. The amide 1H dispersion of 3.6 ppm (6.6-10.2 ppm) in the 15N-HSQC-spectra of instantly refolded NEP-N confirmed the folded state. This successful instant-refolding of NEP-N has been reported for the first-time and the underlying mechanism has been rationalized through establishing the complete backbone-resonance-assignments of NEP-N at 9.7 M urea-denatured state.

Molecular interaction between human SUMO-I and histone like DNA binding protein of Helicobacter pylori (Hup) investigated by NMR and other biophysical tools.

The proteins secreted by bacteria contribute to immune mediated gastric inflammation and epithelial damage; thus aid bacterial invasion in host tissue, and may also interact with host proteins, conspirating a mechanism against host-immune system. The Histone-like DNA binding protein is one of the most abundant nucleoid-associated proteins in Helicobacter pylori (H. pylori). The protein -referred here as Hup- is also secreted in vitro by H. pylori, thus it may have its role in disease pathogenesis. This is possible only if Hup interact with some human proteins including Small-Ubiquitin-like-Modifier (SUMO) proteins. Studies have established that SUMO-proteins participate in various innate-immune pathways and thus promote an efficient immune response to combat pathogenic infections. Sequence analysis revealed the presence of two SUMO interacting motifs (SIMs) and several positively charged lysine residues on the protein surface of Hup. Additionally, SUMO-proteins epitomize negatively charged surface which confers them the ability to bind to DNA/RNA binding proteins. Based on the presence of SIMs as well as charge complementarity between the proteins, it is legitimate to consider that Hup protein would bind to SUMO-proteins. The present study has been undertaken to establish this interaction for the first time using NMR in combination with ITC and other biophysical techniques.

Molecular cloning and biophysical characterization of CXCL3 chemokine.

CXCL3 is a neutrophil activating chemokine that belongs to GRO subfamily of CXC chemokines. GRO chemokine family comprises of three chemokines GRO α (CXCL1), GROß (CXCL2), and GRO γ (CXCL3), which arose as a result of gene duplication events during the course of chemokine evolution. Although primary sequences of GRO chemokines are highly similar, they performs several protein specific functions in addition to their common property of neutrophil trafficking. However, the molecular basis for their differential functions has not well understood. Although structural details are available for CXCL1 and CXCL2, no such information regarding CXCL3 is available till date. In the present study, we have successfully cloned, expressed, and purified the recombinant CXCL3. Around 15mg/L of pure recombinant CXCL3 protein was obtained. Further, we investigated its functional divergence and biophysical characteristics such as oligomerization, thermal stability and heparin binding etc., and compared all these features with its closest paralog CXCL2. Our studies revealed that, although overall structural and oligomerization features of CXCL3 and CXCL2 are similar, prominent differences were observed in their surface characteristics, thus implicating for a functional divergence.

NMR elucidation of monomer-dimer transition and conformational heterogeneity in histone-like DNA binding protein of Helicobacter pylori.

Helicobacter pylori (H. pylori) colonizes under harsh acidic/oxidative stress conditions of human gastrointestinal tract and can survive there for infinitely longer durations of host life. The bacterium expresses several harbinger proteins to facilitate its persistent colonization under such conditions. One such protein in H. pylori is histone-like DNA binding protein (Hup), which in its homo-dimeric form binds to DNA to perform various DNA dependent cellular activities. Further, it also plays an important role in protecting the genomic DNA from oxidative stress and acidic denaturation. Legitimately, if the binding of Hup to DNA is suppressed, it will directly impact on the survival of the bacterium, thus making Hup a potential therapeutic target for developing new anti-H. pylori agents. However, to inhibit the binding of Hup to DNA, it is necessary to gain detailed insights into the molecular and structural basis of Hup-dimerization and its binding mechanism to DNA. As a first step in this direction, we report here the nuclear magnetic resonance (NMR) assignments and structural features of Hup at pH 6.0. The study revealed the occurrence of dynamic equilibrium between its monomer and dimer conformations. The dynamic equilibrium was found to shifting towards dimer both at low temperature and low pH; whereas DNA binding studies evidenced that the protein binds to DNA in its dimeric form. These preliminary investigations correlate very well with the diverse functionality of protein and will form the basis for future studies aiming to develop novel anti-H. pylori agents employing structure-based-rational drug discovery approach.

Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA.

Overexpression of domains of a human protein using recombinant DNA technology has been challenging because individual domains intend to accumulate as non-soluble aggregate when expressed separately. Studies on identifying right sequences for a domain to be able to fold independently may help understand the folding pattern and underlying protein-engineering events to isolate the functional domains of a protein. In this report, individual domains of prostate cancer related biomarkers; MSMB and PSA were overexpressed in bacterial system and purified in their folded forms using affinity chromatography. The western blotting experiment using domain specific antibodies further confirmed these proteins. The designed nucleotide sequences domains were truncated using fold index software and folding were predicted by phyre2 and I-TASSER software. Other parameters were optimized for their overexpression and purification using Co-NTA affinity chromatography. Purified domains of each protein showed secondary structures such as α + ß type for PSA, α/ß and ß type for the each domains of PSA and MSMB respectively. This is the first report on producing PSA and MSMB individual domains in functional folded forms. This study may help produce the folded domain of many such proteins to be used for better diagnostic purpose.