Major Groove Of DNA Research Articles

DNA Deformation Exerted by Regulatory DNA-Binding Motifs in Human Alkyladenine DNA Glycosylase Promotes Base Flipping.

Human alkyladenine DNA glycosylase (AAG) is a key enzyme that corrects a broad range of alkylated and deaminated nucleobases to maintain genomic integrity. When encountering the lesions, AAG adopts a base-flipping strategy to extrude the target base from the DNA duplex to its active site, thereby cleaving the glycosidic bond. Despite its functional importance, the detailed mechanism of such base extrusion and how AAG distinguishes the lesions from an excess of normal bases both remain elusive. Here, through the Markov state model constructed on extensive all-atom molecular dynamics simulations, we find that the alkylated nucleobase (N3-methyladenine, 3MeA) everts through the DNA major groove. Two key AAG motifs, the intercalation and E131-N146 motifs, play active roles in bending/pressing the DNA backbone and widening the DNA minor groove during 3MeA eversion. In particular, the intercalated residue Y162 is involved in buckling the target site at the early stage of 3MeA eversion. Our traveling-salesman based automated path searching algorithm further revealed that a non-target normal adenine tends to be trapped in an exo site near the active site, which however barely exists for a target base 3MeA. Collectively, these results suggest that the Markov state model combined with traveling-salesman based automated path searching acts as a promising approach for studying complex conformational changes of biomolecules and dissecting the elaborate mechanism of target recognition by this unique enzyme.

Synthesis and characterization of single‐walled carbon nanotube: Cyto‐genotoxicity in Allium cepa root tips and molecular docking studies

Herein, single-walled carbon nanotubes (SWCNTs) were synthesized by the thermal chemical vapor deposition (CVD) method, and characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), Raman spectroscopy, dynamic light scattering (DLS), and thermo-gravimetric analysis (TGA). The results indicated that obtained nanotubes were SWCNTs with high crystallinity and their average diameter was 10.15 ± 3nm. Allium cepa ana-telophase and comet assays on the root meristem were employed to evaluate the cytotoxic and genotoxic effects of SWCNTs by examining mitotic phases, mitotic index (MI), chromosomal aberrations (CAs), and DNA damage. A. cepa root tip cells were exposed to SWCNTs at concentrations of 12.5, 25, 50, and 100 μg/ml for 4h. Distilled water and methyl methanesulfonate (MMS, 10μg/ml) were used as the negative and positive control groups, respectively. It was observed that MIs decreased statistically significantly for all applied doses. Besides, CAs such as chromosome laggards, disturbed anaphase-telophase, stickiness and bridges and also DNA damage increased in the presence of SWCNTs in a concentration-dependent manner. In the molecular docking study, the SWCNT were found to be a strong DNA major groove binder showing an energetically very favorable binding free energy of -21.27 kcal/mol. Furthermore, the SWCNT interacted effectively with the nucleotides on both strands of DNA primarily via hydrophobic π and electrostatic interactions. As a result, cytotoxic and genotoxic effects of SWCNTs in A. cepa root meristematic cells which is a reliable system for assessment of nanoparticle toxicology were demonstrated in this study.

Synthesis of water soluble symmetric and asymmetric pillar[5]arene derivatives: Cytotoxicity, apoptosis and molecular docking studies

In this study, two water-soluble pillar[5]arenes, [1-(3-(trimethyl-azane)propoxy)-4-methoxyhydro-chloridepillar[5]arene (asym-P5) and 1,4-bis(2-(trimethyl-azane)ethoxy)hydrobromidepillar[5]arene (sym-P5), were synthesized and characterized using fourier transform infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (1H-NMR and 13C-NMR). Cytotoxic and apoptotic effects of asym-P5 and sym-P5 on human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MDA-MB-231), human colon carcinoma (Caco-2), human prostate cancer (LnCap) and human embryonic kidney (HEK293) cell lines were determined by MTT, Annexin V-FITC apoptosis assays and quantitative analysis of mRNA levels of apoptosis related genes (Bax, Bcl-2, caspase 3, 8, and 9), respectively. asym-P5 not only reduced cell viability in all cell lines in a dose-dependent manner but also increased the apoptotic cell percentage significantly. sym-P5 was less effective than asym-P5 in inducing these effects in cancer cell lines. In RT-qPCR studies, it was observed that after asym-P5 treatment, Bax and caspase-3 mRNA levels increased, however, Bcl-2 levels decreased compared to the control group. As a result of the molecular docking, asym-P5 exhibited an energetically more favorable DNA binding affinity (-5.4 kcal/mol) than sym-P5 (-4.5 kcal/mol), where both ligands conformationally snug-fit into the major groove of DNA. These results demonstrated that asym-P5 had cytotoxic and apoptotic effects in cell lines.

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Tetramethyl-phenanthroline copper complexes in the development of drugs to treat cancer: synthesis, characterization and cytotoxicity studies of a series of copper(II)-L-dipeptide-3,4,7,8-tetramethyl-phenanthroline complexes.

New compounds to fight cancer are needed due to cancer high incidence and lack of curative treatments for several classes of this disease. Metal-based coordination compounds offer a variety of molecules that can turn into drugs. Among them, coordination copper complexes are emerging as an attractive class of compounds for cancer treatment. A series of [Cu(L-dipeptide)(tmp)] (tmp = 3,4,7,8-tetramethyl-1,10-phenanthroline) complexes were synthesized and characterized in the solid state, including the determination of the crystalline structure of [Cu(Gly-Gly)(tmp)]·3.5 H2O and [Cu2Cl4(tmp)2]. The complexes were studied in solution, where the major species are also ternary ones. The lipophilicity of the complexes was determined and the binding to the DNA was evaluated, suggesting that it occurs in the DNA's major groove. The cytotoxicity of the complexes was evaluated on different cancer cell lines: human metastatic breast adenocarcinoma MDA-MB-231 (triple negative, ATCC: HTB-26), MCF-7 (ATCC: HTB-22),SK-BR-3 (ATCC: HTB-30),human lung epithelial carcinomaA549(ATCC: CCL-185),cisplatin resistant-human ovarian carcinomaA2780cis (SIGMA)and nontumoral cell lines: MRC-5 (lung; ATCC: CCL-171) and MCF-10A (breast, ATCC: CRL-10317). [Cu(L-dipeptide)(tmp)] complexes are highly cytotoxic as compared to [Cu(L-dipeptide)(phenanthroline)] and cisplatin. Therefore, [Cu(L-dipeptide)(tmp)] complexes are promising candidates to have their in vivo activity further studied toward new treatments for triple negative breast cancer and other aggressive tumors for which there is no curative pharmacological treatment to the date.

Role of key protein residues involved in DinB DNA damage specificity: an in silico study

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb, and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little finger” or polymerase‐associated domain (PAD) and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. Specifically, we analyzed a lesion that the protein can bypass (N2‐furfuryl‐dG) and one that instead the protein does not bypass well (O6‐Methylated dG). Our MD simulations revealed that there are some key residues in the fingers domain that are important switches for the conformational change. For example, R35 assumes a characteristic position when the protein is in a catalytically competent state, while it blocks the active site when the protein is not able to bypass the lesion. This result is in agreement with previous studies which showed that mutations of the R35 residue alter the protein specificity. Other areas of DinB which seem to undergo large movement reside in the PAD domain. This domain, which makes contact with the DNA major groove, has been implicated in DNA damage specificity, so it makes sense that residues on this domain assume different conformations, depending on the type of lesions present on the templating strand. A discussion of possible roles for some of the residues on the PAD will be presented.

A muconate bridged bipyridyl appended binuclear Cu(II) complex reveals dissimilar affinities to DNA and BSA protein

A new muconate based Cu(II) metal complex, [Cu2(muco)(5,5′-Me2bpy)4]·2ClO4¯ (1; H2muco = trans, trans-1,3-butadiene-1,4-dicarboxylic acid or trans, trans-muconic acid and 5,5′-Me2bpy = 5,5′-dimethyl-2,2′-bipyridine), has been synthesized and structurally characterized. X-ray structure analysis reveals that 1 forms a binuclear complex with a five coordinated square pyramidal geometry of the central Cu(II) ion attached to a muco and two bipyridine ligands. Docking studies have been carried out which reveal the successful binding of the metal complex to the major groove of DNA and the planar aromatic bipyridyl ligand partially intercalates into the base pairs. The synthesized complex fits into the pocket of bovine serum albumin (BSA), while the maximum fraction submerges into the BSA binding pocket. An extra coordinate bond has been found, forming through the oxygen atom of 158 asparagines amino acid, which subsequently forms a new six coordinated octahedral complex. Experimental studies have been performed to validate all the data obtained from the docking studies. This study reveals highly diverse affinities and binding modes of the synthesized complex towards DNA and protein. Therefore, the synthesized complex may pave the way to be used as a specific protein binder (low affinity to DNA or RNA) bioactive compound.

Selective and stable base pairing by alkynylated nucleosides featuring a spatially-separated recognition interface

Unnatural base pairs (UBPs) which exhibit a selectivity against pairing with canonical nucleobases provide a powerful tool for the development of nucleic acid-based technologies. As an alternative strategy to the conventional UBP designs, which involve utility of different recognition modes at the Watson–Crick interface, we now report that the exclusive base pairing can be achieved through the spatial separation of recognition units. The design concept was demonstrated with the alkynylated purine (NPu, OPu) and pyridazine (NPz, OPz) nucleosides endowed with nucleobase-like 2-aminopyrimidine or 2-pyridone (‘pseudo-nucleobases’) on their major groove side. These alkynylated purines and pyridazines exhibited exclusive and stable pairing properties by the formation of complementary hydrogen bonds between the pseudo-nucleobases in the DNA major groove as revealed by comprehensive Tm measurements, 2D-NMR analyses, and MD simulations. Moreover, the alkynylated purine-pyridazine pairs enabled dramatic stabilization of the DNA duplex upon consecutive incorporation while maintaining a high sequence-specificity. The present study showcases the separation of the recognition interface as a promising strategy for developing new types of UBPs.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

RpoN-Based stapled peptides with improved DNA binding suppress Pseudomonas aeruginosa virulence.

Stapled peptides have the ability to mimic α-helices involved in protein binding and have proved to be effective pharmacological agents for disrupting protein-protein interactions. DNA-binding proteins such as transcription factors bind their cognate DNA sequences via an α-helix interacting with the major groove of DNA. We previously developed a stapled peptide based on the bacterial alternative sigma factor RpoN capable of binding the RpoN DNA promoter sequence and inhibiting RpoN-mediated expression in Escherichia coli. We have elucidated a structure-activity relationship for DNA binding by this stapled peptide, improving DNA binding affinity constants in the high nM range. Lead peptides were shown to have low toxicity as determined by their low hemolytic activity at 100 μM and were shown to have anti-virulence activity in a Galleria mellonella model of Pseudomonas aeruginosa infection. These findings support further preclinical development of stapled peptides as antivirulence agents targeting P. aeruginosa.

Crystal structure of the BREX phage defence protein BrxA.

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multitudinous and varied resistance mechanisms. Bacteriophage Exclusion (BREX) systems protect from phage infection by generating methylation patterns at non-palindromic 6 bp sites in host bacterial DNA, to distinguish and block replication of non-self DNA. Type 1 BREX systems are comprised of six conserved core genes. Here, we present the first reported structure of a BREX core protein, BrxA from the phage defence island of Escherichia fergusonii ATCC 35469 plasmid pEFER, solved to 2.09 ​Å. BrxA is a monomeric protein in solution, with an all α-helical globular fold. Conservation of surface charges and structural homology modelling against known phage defence systems highlighted that BrxA contains two helix-turn-helix motifs, juxtaposed by 180°, positioned to bind opposite sides of a DNA major groove. BrxA was subsequently shown to bind dsDNA. This new understanding of BrxA structure, and first indication of BrxA biological activity, suggests a conserved mode of DNA-recognition has become widespread and implemented by diverse phage defence systems.

Structural specificity of groove binding mechanism between imidazolium-based ionic liquids and DNA revealed by synchrotron-UV Resonance Raman spectroscopy and molecular dynamics simulations

The predicted capability of Ionic Liquids (ILs) in stabilizing the native structure of nucleic acids is relevant in biotechnology, especially for DNA storage and handling. In the present work, we implement a joint combination of advanced spectroscopic techniques such as synchrotron radiation-UV Resonance Raman spectroscopy (SR-UVRR) and molecular dynamics (MD) simulations for deepening insight into the sequence and structural specificity of the binding interactions between imidazolium-based ILs and both the phosphate groups and nucleobases in the minor and major grooves of double-stranded DNA. A 30-base pair double-stranded DNA structure has been chosen as a model of natural DNA. The experimental and simulation results give evidence of the predominance of a groove binding mechanism between ILs cations and DNA, with preferential interactions among guanine residues and the shorter alkyl-chain length on imidazolium cations. Raman experiments allowed us to detect both cooperative transition and reversible pre-melting structural transformations that involve specific tracts in the structure of DNA and are turned on at lower temperatures for guanine residues than for adenine ones. The more marked effect on the pre-melting states of adenine operated by the imidazolium-based ILs with chloride as anion suggests a selective strong interaction of this anion with the DNA’s adenine-rich tracts. MD simulation results reveal the influence of ILs on the structural properties of DNA and provide more details about the solvation, interaction, stability and flexibility of DNA in the hydrated ILs. According to MD analyses, simultaneous electrostatic and hydrophobic interactions drive the shorter alkyl-chain length of imidazolium cations to have greater interplays with the DNA major groove.

DNA binding graphene quantum dots inhibit dual topoisomerases for cancer chemotherapy

DNA-protein interfacial inhibition is a classical paradigm for design of diverse interfacial inhibitors as clinically relevant anticancer drugs. To extend this paradigm from small molecules to nanoparticles, we synthesized pyridine N-modified polycationic graphene quantum dots (Graphenin) as a new class of DNA probes and interfacial inhibitors for cancer chemotherapy. Compared with classical interfacial inhibitors, Graphenin showed enhanced interfacial inhibition against Topoisomerase I and II, both of which are clinically important targets for chemotherapy. The enhanced interfacial inhibition activity was ascribed to their distinct DNA targeting capability preferentially at the DNA major groove, through which many DNA binding proteins recognize DNA. They induced DNA damage and cell apoptosis for sensitive and resistant cancer cell lines, and great potential for tumor chemotherapy was demonstrated in vitro and in vivo. Our results have identified shape-complementary Graphenin as the first DNA major groove binder to inhibit DNA binding enzymes for chemotherapy.

Structural basis of the complete poxvirus transcription initiation process.

Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

Lead (Pb) induced Oxidative Stress as a Mechanism to Cause Neurotoxicity in Drosophila melanogaster

The widespread use of lead (Pb) has caused global contamination, inevitable human exposure, and public health problems. Pb neurotoxicity has been linked to various human diseases, but its associated mechanism causing neurotoxicity is unknown. Drosophila melanogaster as a model organism has been used to study the mechanism involved in Pb-caused neurotoxicity and the potential role of antioxidants in ameliorating its harmful effects. The larval feeding technique was adopted to administer different concentrations of Pb (0.2-0.8 mM) to Oregon-R (ORR), superoxide dismutase (Sod), or catalase (Cat) overexpressing, and Sod or Cat knockdown flies to analyse Pb load, oxidative stress components, DNA damage, apoptosis and vacuolation in the brain. The results revealed that Pb accumulation in the Drosophila brain induces oxidative stress by generating reactive oxygen species (ROS) and lipid peroxidation (LPO), depleting antioxidant enzymes. Molecular docking studies have evidenced it. Pb directly binds to antioxidants and major grooves of DNA, leading to DNA damage. Increased DNA damage, apoptosis, vacuolation in brains of Pb-treated ORR, Sod, or Cat knockdown flies; and on the contrary, reduced oxidative DNA damage, apoptosis, and vacuolation in brains of Pb treated Sod or Cat overexpressed flies put forward that oxidative stress is the mechanism in Pb caused neurotoxicity.

Supramolecular Assembly of a Terpyridyl based Binuclear Cu(II) Complex and its DNA Docking Study

ABSTRACT A terpyridyl-based binuclear Cu(II) complex, formulated as [Cu2(tpy)2(muco)(NO3)2], (1) (typ = 2,2’;6’,2”-terpyridine and H2-muco = trans, trans-muconic acid) was synthesised in a single pot technique using slow evaporation method. Blue coloured distorted octahedral-shaped crystals were obtained after a day. Synthesised compound has been characterised by elemental analysis as well as single crystal X-ray diffraction (SCXRD) techniques. Hirshfeld surface analysis has been explored to check out the nature of intermolecular interactions and packing modes of compounds in the crystalline state. From the docking study, it has been found that the metal complex bind to the major groove of DNA and the planner aromatic pyridine ring of the ligand partially intercalate into the base pairs.

Synthesis, characterization, antimicrobial, cytotoxic, DNA-interaction, molecular docking and DFT studies of novel di- and tri-organotin(IV) carboxylates using 3-(3-nitrophenyl)2-methylpropenoic acid

One diorganotin(IV) [n-Bu2SnL2] (1) and two triorganotin(IV) carboxylates [n-Bu3SnL] (2) and [Ph3SnL] (3) [L = 3-(3-nitrophenyl) 2-methylpropenoic acid] were synthesized and characterized by elemental analysis, FT-IR and NMR (1H, 13C) spectroscopies. The geometry of complexes and binding mode of ligand were worked out through FT-IR and NMR spectroscopies. The ligand coordinate with Sn via carboxylato oxygen in monodentate fashion leading to four coordinated geometries around Sn center. The effectiveness of complexes towards their antimicrobial and cytotoxic potential was evaluated and a significant extent of antimicrobial potential was observed with a few exceptions. Molecular docking studies were performed for these complexes to check their interaction with DNA. Results from this study revealed that these compounds can bind favorably with cisplatin binding site and targeting the major groove of DNA. The complex-DNA interaction study was also performed through UV-Vis spectroscopic technique and viscosity measurement, and the observed experimental results were well matched with theoretical results. Computational vibrational analysis, frontier molecular orbital (FMO), natural bond orbitals (NBOs), linear and non-linear optical (NLO) properties of ligand and complexes 1-3 were calculated by density functional theory (DFT) and time-dependent DFT (TDDFT) using CAM-B3LYP/6-31G (d,p) level of theory to evaluate spectroscopic, structural parameters and reactivity patterns.

Non-flipping DNA glycosylase AlkD scans DNA without formation of a stable interrogation complex

The multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. While DNA glycosylases in the BER pathway generally bend the DNA and flip damaged bases into lesion specific pockets, the HEAT-like repeat DNA glycosylase AlkD detects and excises bases without sequestering the base from the DNA helix. We show by single-molecule tracking experiments that AlkD scans DNA without forming a stable interrogation complex. This contrasts with previously studied repair enzymes that need to flip bases into lesion-recognition pockets and form stable interrogation complexes. Moreover, we show by design of a loss-of-function mutant that the bimodality in scanning observed for the structural homologue AlkF is due to a key structural differentiator between AlkD and AlkF; a positively charged β-hairpin able to protrude into the major groove of DNA.

Novel DNA Bis-Intercalator XR5944 as a Potent Anticancer Drug—Design and Mechanism of Action

This review is dedicated to Professor William A. Denny’s discovery of XR5944 (also known as MLN944). XR5944 is a DNA-targeted agent with exceptionally potent antitumor activity and a novel DNA binding mode, bis-intercalation and major groove binding, as well as a novel mechanism of action, transcription inhibition. This novel anticancer compound represents a remarkable accomplishment resulting from two decades of drug discovery by Professor Denny and coworkers. Here, we review our work on the structural study of the DNA binding mode of XR5944 and mechanistic study of XR5944 action.

Stereochemical Trajectories of a Two-Component Regulatory System PmrA/B in a Colistin-Resistant Acinetobacter baumannii Clinical Isolate

Background:There is limited information on the 3D prediction and modeling of the colistin resistance-associated proteins PmrA/B TCS in Acinetobacter baumannii. We aimed to evaluate the stereochemical structure and domain characterization of PmrA/B in an A. baumannii isolate resistant to high-level colistin, using bioinformatics tools. Methods:The species of the isolate and its susceptibility to colistin were confirmed by PCR-sequencing and MIC assay, respectively. For 3D prediction of the PmrA/B, we used 16 template models with the highest quality (e-value <1 × 10−50). Results:Prediction of the PmrA structure revealed a monomeric non-redundant protein consisting of 28 α-helices and 22 β-sheets. The PmrA DNA-binding motif displayed three antiparallel α-helices, followed by three β-sheets, and was bond to the major groove of DNA by intermolecular van der Waals bonds through amino acids Lys, Asp, His, and Arg, respectively. Superimposition of the deduced PmrA 3D structure with the closely related PmrA protein model (GenBank no. WP_071210493.1) revealed no distortion in conformation, due to Glu→Lys substitution at position 218. Similarly, the PmrB protein structure displayed 24 α-helices and 13 β-sheets. In our case, His251 acted as a phosphate receptor in the HisKA domain. The amino acid substitutions were mainly observed at the putative N-terminus region of the protein. Furthermore, two substitutions (Lys21→Ser and Ser28→Arg) in the transmembrane domain were detected. Conclusion: TheDNA-binding motif of PmrA is highly conserved, though the N-terminal fragment of PmrB showed a high rate of base substitutions. This research provides valuable insights into the mechanism of colistin resistance in A. baumannii.