In silico identification of drug targets and vaccine candidates against Bartonella quintana: a subtractive proteomics approach.

BACKGROUND: The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts. OBJECTIVE: Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana. METHODS AND FINDINGS: To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively. MAINS CONCLUSIONS: This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.

Unravelling carbohydrate binding module 21 (CBM21) dynamics of interaction with amylose.

The carbohydrate binding module 21 (CBM21) from Rhizopus oryzae is a dual-site CBM proposed to disrupt polysaccharide structures. Additionally, it serves as a purification tag in industry. CBM21 crystal structure features a Glc residue in an unusual 1S3 conformation, whose relevance for the CBM mechanism of action is unclear. In this context, we seek to contribute for the understanding of CBM21 mechanism of action by: i) investigating the role of the 1S3 conformation on carbohydrate recognition, and ii) characterize the protein-carbohydrate binding dynamics using molecular dynamics and metadynamics simulations at MM and QM/MM levels. Results indicate the 1S3 Glc conformation is unlikely to occur under biological conditions, being originated from the crystallographic environment. CBM21 binding to small ligands appears transient and unstable, while protein dimerization and polysaccharide chain size influence complex stability. In interactions with amylose, CBM21 exhibits a repeated unbinding followed by re-binding, while simultaneously alternating between binding sites I and II. These results suggest that CBM21 acts through transient interactions, directing carbohydrates to the catalytic center rather than forming strong and long-lasting bonds with carbohydrates. Accordingly, we expect such atomistic depiction of CBM21 mechanism could aid in CBM design targeting biotechnological applications.

SIRAH Late Harvest: Coarse-Grained Models for Protein Glycosylation.

Glycans constitute one of the most complex families of biological molecules. Despite their crucial role in a plethora of biological processes, they remain largely uncharacterized because of their high complexity. Their intrinsic flexibility and the vast variability associated with the many combination possibilities have hampered their experimental determination. Although theoretical methods have proven to be a valid alternative to the study of glycans, the large size associated with polysaccharides, proteoglycans, and glycolipids poses significant challenges to a fully atomistic description of biologically relevant glycoconjugates. On the other hand, the exquisite dependence on hydrogen bonds to determine glycans' structure makes the development of simplified or coarse-grained (CG) representations extremely challenging. This is particularly the case when glycan representations are expected to be compatible with CG force fields that include several molecular types. We introduce a CG representation able to simulate a wide variety of polysaccharides and common glycosylation motifs in proteins, which is fully compatible with the CG SIRAH force field. Examples of application to N-glycosylated proteins, including antibody recognition and calcium-mediated glycan-protein interactions, highlight the versatility of the enlarged set of CG molecules provided by SIRAH.

Theoretical models of staurosporine and analogs uncover detailed structural information in biological solution.

Staurosporine and its analogs (STA-analogs) are indolocarbazoles (ICZs) compounds able to inhibit kinase proteins in a non-specific way, while present antimicrobial and cytostatic properties. The knowledge of molecular features associated to the complexation, including the ligand shape in solution and thermodynamics of complexation, is substantial to the development of new bioactive ICZs with improved therapeutic properties. In this context, the empirical approach of GROMOS force field is able to accurately reproduce condensed phase physicochemical properties of molecular systems after parameterization. Hence, through parameterization under GROMOS force field and molecular simulations, we assessed STA-analogs dynamics in aqueous solution, as well as its interaction with water to probe conformational and structural features involved in complexation to therapeutic targets. The coexistence of multiple conformers observed in simulations, and confirmed by metadynamics calculations, expanding the conformational space knowledge of these ligands with potential implications in understanding the ligand conformational selection during complexation. Also, changes in availability to H-bonding concerning the different substituents and water can reflect on effects at complexation free energy due to variation at the desolvation energetic costs. Based on these results, we expect the obtained structural data provide systemic framework for rational chemical modification of STA-analogs.

Capparis cartilaginea decne (capparaceae): isolation of flavonoids by high-speed countercurrent chromatography and their anti-inflammatory evaluation.

Introduction: Capparis cartilaginea Decne. (CC) originates from the dry regions of Asia and the Mediterranean basin. In traditional medicine, tea of CC leaves is commonly used to treat inflammatory conditions such as rheumatism, arthritis, and gout. Due to the limited studies on the phytochemistry and biological activity of CC compared to other members of the Capparaceae family, this work aims to: 1) Identify the chemical composition of CC extract and 2) Investigate the potential anti-inflammatory effect of CC extract, tea and the isolated compounds. Methods: To guarantee aim 1, high-speed countercurrent chromatography (HSCC) method; Nuclear Magnetic Resonance (NMR) and High-Performance Liquid Chromatography coupled to Electrospray Ionisation and Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESIQTOF-MS/MS) were employed for this purpose. To guarantee aim 2, we studied the effect of the isolated flavonoids on matrix metalloproteinases (MMPs) -9 and -2 in murine macrophages. Molecular docking was initially performed to assess the binding affinity of the isolated flavonoids to the active site of MMP-9. Results and discussion: In silico model was a powerful tool to predict the compounds that could strongly bind and inhibit MMPs. CC extract and tea have shown to possess a significant antioxidant and anti-inflammatory effect, which can partially explain their traditional medicinal use.

Revisiting the structural basis for biological activity of GMI-1070, a sialyl Lewisx mimetic.

When it comes to the treatment of pathologies in which aberrant cell adhesion and extravasation from the bloodstream have been implicated, the selectins represent a central therapeutic target. In this context, the present work investigates the conformational landscape of two prototypes for the design of new antineoplasic and anti-inflammatory agents: the natural selectin ligand sialyl Lewisx and its mimetic GMI-1070. Accordingly, a series of unbiased molecular dynamics simulations at the microsecond scale using GROMOS 53A6 (GLYC), CHARMM36m and GLYCAM06 force fields were employed, together with ConfID, an analytical method for the characterization of conformational populations of small molecules. Our results for sialyl Lewisx are in agreement with and expand upon prior work. As for the mimetic, our results indicate that, in spite of its conformational restriction, GMI-1070's behavior in solution deviates from what had been proposed, highlighting thus some features that could be optimized, as the development of sialyl Lewisx mimetics continues, and new candidates emerge.

L-proline transporter inhibitor (LQFM215) promotes neuroprotection in ischemic stroke.

BACKGROUND: L-proline transporter (PROT/SLC6A7) is closely associated with glutamatergic neurotransmission, where L-proline modulates the NMDA receptor (NMDAR) function. NMDAR-mediated excitotoxicity is a primary cause of neuronal death following stroke, which is triggered by the uncontrolled release of glutamate during the ischemic process. After ischemic stroke, L-proline levels show a reduction in the plasma, but high circulating levels of this molecule indicate good functional recovery. This work aimed to produce new PROT inhibitors and explore their effects on ischemic stroke. METHODS: Initially, we built a three-dimensional model of the PROT protein and run a molecular docking with the newly designed compounds (LQFM215, LQFM216, and LQFM217). Then, we synthesized new PROT inhibitors by molecular hybridization, and proline uptake was measured in ex vivo and in vivo models. The behavioral characterization of the treated mice was performed by the open-field test, elevated plus-maze, Y-maze, and forced swimming test. We used the permanent middle cerebral artery occlusion (MCAO) model to study the ischemic stroke damage and analyzed the motor impairment with limb clasping or cylinder tests. RESULTS: LQFM215 inhibited proline uptake in hippocampal synaptosomes, and the LQFM215 treatment reduced proline levels in the mouse hippocampus. LQFM215 reduced the locomotor and exploratory activity in mice and did not show any anxiety-related or working memory impairments. In the MCAO model, LQFM215 pre-treatment and treatment reduced the infarcted area and reduced motor impairments in the cylinder test and limb clasping. CONCLUSIONS: This dataset suggests that the new compounds inhibit cerebral L-proline uptake and that LQFM215 promotes neuroprotection and neuro-repair in the acute ischemic stroke model.

Structural Characteristics of Glycocins: Unraveling the Role of S-Linked Carbohydrates and Other Structural Elements.

Glycocins are antimicrobial peptides with glycosylations, often an S-linked monosaccharide. Their recent structure elucidation has brought forth questions about their mechanisms of action as well as the impact of S-glycosylation on their structural behavior. Here, we investigated structural characteristics of glycocins using a computational approach. Depending on the peptide's class (sublancin- or glycocin F-like), the sugar changes the peptide's flexibility. Also, the presence of glycosylation is necessary for the lack of structure of Asm1. The C-terminal tail in glycocin F-like peptides influenced their structured regions, acting like a regulator. These findings corroborate the versatility of these post-translational modifications, pointing toward their potential use in molecular engineering.

Accommodation of In-Register N-Linked Glycans on Prion Protein Amyloid Cores.

Although prion protein fibrils can have either parallel-in-register intermolecular ß-sheet (PIRIBS) or, probably, ß-solenoid architectures, the plausibility of PIRIBS architectures for the usually glycosylated natural prion strains has been questioned based the expectation that such glycans would not fit if stacked in-register on each monomer within a fibril. To directly assess this issue, we have added N-linked glycans to a recently reported cryo-electron microscopy-based human prion protein amyloid model with a PIRIBS architecture and performed in silico molecular dynamics studies to determine if the glycans can fit. Our results show that triantennary glycans can be sterically accommodated in-register on both N-linked glycosylation sites of each monomer. Additional simulations with an artificially mutated ß-solenoid model confirmed that glycans can be accommodated when aligned with ∼4.8 Å spacing on every rung of a fibril. Altogether, we conclude that steric intermolecular clashes between glycans do not, in themselves, preclude PIRIBS architectures for prions.

Rotational Profiler: A Fast, Automated, and Interactive Server to Derive Torsional Dihedral Potentials for Classical Molecular Simulations.

Rotational Profiler provides an analytical algorithm to compute sets of classical torsional dihedral parameters by fitting an empirical energy profile to a reference one that can be obtained experimentally or by quantum-mechanical methods. The resulting profiles are compatible with the functional forms in the most widely used biomolecular force fields (e.g., GROMOS, AMBER, OPLS, and CHARMM). The linear least-squares regression method is used to generate sets of parameters that best satisfy the fitting. Rotational Profiler is free to use, analytical, and force field/package independent. The formalism is herein described, and its usage, in an interactive and automated manner, is made available as a Web server at http://rotprof.lncc.br.

Neuropharmacological assessment in mice and molecular docking of piperazine derivative LQFM212.

Piperazine derivatives are an attractive class of chemical compounds for the treatment of various mental illness. Herein, we demonstrated the synthesis of LQFM212, a piperazine derivative, behavioral evaluation in mice and computational studies. In neuropharmacological assessment, LQFM212 treatment at doses of 18, 54 or 162 µmol/kg increased the sleep duration in sodium pentobarbital-induced sleep test. LQFM212 at dose of 162 µmol/kg increased climbing time in the chimney test and decreased the number of squares crossed in the open field test, suggesting that LQFM212 in high doses reduces spontaneous movement. However, LQFM212 treatment at the doses of 18 or 54 µmol/kg increased the preference for the center of field which could be indicative of anxiolytic-like effects. In elevated plus maze and light-dark box tests, LQFM212 treatment altered all parameters observed that demonstrate anxiolytic-like activity. These effects were reversed by flumazenil, mecamylamine, WAY-100635 and PCPA, but not with ketanserin, showing that anxiolytic-like activity involve benzodiazepine site of GABAA receptor, nicotinic and serotonergic pathways. Molecular docking of LQFM212 showed that the ligand has more interactions with GABAA receptor than with 5-HT1A receptor. Despite the involvement of benzodiazepine site on anxiolytic-like effect of LQFM212, treatment with this compound did not alter cognitive function in the step-down avoidance test. In this sense, this piperazine derivative is a good prototype for treating anxiety disorders with putative mechanism of action.

Design, synthesis and pharmacological assessment of new pyrazole compounds.

AIMS: This study investigated the antinociceptive and anti-inflammatory effects of new pyrazole compounds LQFM011(5), LQFM043(6) and LQFM044(7) as well as the mechanisms of action and acute in vitro toxicity. MAIN METHODS: The antinociceptive activity was evaluated using the acetic acid-induced abdominal writhing test, formalin-induced pain test and the Randall-Selitto test. The anti-inflammatory activity was evaluated using models of paw oedema and pleurisy induced by carrageenan; cell migration, the levels of tumour necrosis factor α (TNF-α) and myeloperoxidase (MPO) enzyme activity were evaluated. In addition, the ability to inhibit phospholipase A2 (PLA2) in vitro and docking in PLA2 were used. Acute oral systemic toxicity in mice was evaluated through the neutral red uptake assay. KEY FINDINGS: The synthesised compounds (5-7), delivered via gavage (p.o.) at 70, 140 or 280 µmol/kg, decreased the number of writhings induced by acetic acid; the three compounds (280 µmol/kg p.o.) reduced the paw licking time in the first and second phase of the formalin test and decreased the nociceptive threshold variation in the Randall-Selitto test. Furthermore, this dose reduced oedema formation, leucocyte migration (specifically through reduction in polymorphonuclear cell movement) and increased mononuclear cells. MPO activity and the levels of pro-inflammatory cytokines TNF-α were decreased. Evaluation of PLA2 inhibition via the docking simulation revealed more interactions of LQFM043R(6) and LQFM044(7), data that corroborated the half-maximal inhibitory concentration (IC50) of PLA2 inhibition in vitro. Therefore, LQFM011(5), LQFM043(6) and LQFM044(7) were classified with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) as category 4.

Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes.

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

A cell surface arabinogalactan-peptide influences root hair cell fate.

Root hairs (RHs) develop from specialized epidermal trichoblast cells, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling RH cell fate is only partially understood. RH cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE-4 (RSL4) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence RH cell fate. In the absence of BRs, phosphorylated BIN2 (a Type-II GSK3-like kinase) inhibits a protein complex that regulates GL2 expression. Perturbation of the arabinogalactan peptide (AGP21) in Arabidopsis thaliana triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O-glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2-dependent manner. Changes in cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermis.

ConfID: an analytical method for conformational characterization of small molecules using molecular dynamics trajectories.

MOTIVATION: The conformational space of small molecules can be vast and difficult to assess. Molecular dynamics (MD) simulations of free ligands in solution have been applied to predict conformational populations, but their characterization is often based on clustering algorithms or manual efforts. RESULTS: Here, we introduce ConfID, an analytical tool for conformational characterization of small molecules using MD trajectories. The evolution of conformational sampling and population frequencies throughout trajectories is calculated to check for sampling convergence while allowing to map relevant conformational transitions. The tool is designed to track conformational transition events and calculate time-dependent properties for each conformational population detected. AVAILABILITY AND IMPLEMENTATION: Toolkit and documentation are freely available at http://sbcb.inf.ufrgs.br/confid. CONTACT: marcelo.poleto@ufv.br or bigrisci@inf.ufrgs.br. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Current Status of Carbohydrates Information in the Protein Data Bank.

Carbohydrates are well known for their physicochemical, biological, functional, and therapeutic characteristics. Unfortunately, their chemical nature imposes severe challenges for the structural elucidation of these phenomena, impairing not only the depth of our understanding of carbohydrates but also the development of new biotechnological and therapeutic applications based on these molecules. In the recent past, the amount of structural information, obtained mainly from X-ray crystallography, has increased progressively, as well as its quality. In this context, the current work presents a global analysis of the carbohydrate information available in the Protein Data Bank (PDB). From high quality structures, it is clear that most of the data are highly concentrated on a few sets of residue types, on their monosaccharidic forms, and connected by a small diversity of glycosidic linkages. The geometries of these linkages can be mostly associated with the types of linkages instead of residues, while the level of puckering distortion was characterized, quantified, and located in a pseudorotational equilibrium landscape, not only to local minima but also to transitional states. These qualitative and quantitative analyses offer a global picture of the carbohydrate structural content in the PDB, potentially supporting the building of new models for carbohydrate-related biological phenomena at the atomistic level, including new developments on force field parameters.

The Lazy Life of Lipid-Linked Oligosaccharides in All Life Domains.

Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein.

Novel choline analog 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol produces sympathoinhibition, hypotension, and antihypertensive effects.

The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2 mg/kg) was administered by intravenous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels, while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032 elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective muscarinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) attenuated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of muscarinic receptors.

Development of GROMOS-Compatible Parameter Set for Simulations of Chalcones and Flavonoids.

Chalcones and flavonoids constitute a large family of plant secondary metabolites that have been explored as a potential source of novel pharmaceutical products. While the simulation of these compounds by molecular dynamics (MD) can be a valuable strategy to assess their conformational properties and so further develop their role in drug discovery, there are no set of force field parameters specifically designed and experimentally validated for their conformational description in condensed phase. So the current work developed a new parameter set for MD simulations of these compounds' main scaffolds under GROMOS force field. We employed a protocol adjusting the atomic charges and torsional parameters to the respective quantum mechanical derived dipole moments and dihedrals rotational profiles, respectively. Experimental properties of organic liquids were used as references to the calculated values to validate the parameters. Additionally, metadynamics simulations were performed to evaluate the conformational space of complex chalcones and flavonoids, while NOE contacts during simulations were measured and compared to experimental data. Accordingly, the employed protocol allowed us to obtain force field parameters that reproduce well the target data and may be expected to contribute in more accurate computational studies on the biological/therapeutical role of such molecules.

Dynamics of DDB2-DDB1 complex under different naturally-occurring mutants in Xeroderma Pigmentosum disease.

BACKGROUND: Xeroderma Pigmentosum (XP) is a disease caused by mutations in the nucleotide excision repair (NER) pathway. Patients with XP exhibit a high propensity to skin cancers and some subtypes of XP can even present neurological impairments. During NER, DDB2 (XPE), in complex with DDB1 (DDB-Complex), performs the DNA lesion recognition. However, not much is known about how mutations found in XP patients affect the DDB2 structure and complex assembly. Thus, we searched for structural evidence associated with the role of three naturally occurring mutations found in XPE patients: R273H, K244E, and L350P. METHODS: Each mutant was individually constructed and submitted to multiple molecular dynamics simulations, done in triplicate for each designed system. Additionally, Dynamic Residue Interaction Networks were designed for each system and analyzed parallel with the simulations. RESULTS: DDB2 mutations promoted loss of flexibility in the overall protein structure, producing a different conformational behavior in comparison to the WT, especially in the region comprising residues 354 to 371. Furthermore, the DDB-complex containing the mutated forms of DDB2 showed distinct behaviors for each mutant: R273H displayed higher structural instability when complexed; L350P affected DDB1 protein-protein binding with DDB2; and K244E, altered the complex binding trough different ways than L350P. CONCLUSIONS: The data gathered throughout the analyses helps to enlighten the structural basis for how naturally occurring mutations found in XPE patients impact on DDB2 and DDB1 function. GENERAL SIGNIFICANCE: Our data influence not only on the knowledge of XP but on the DNA repair mechanisms of NER itself.