Unbound Form Research Articles

Cryo-EM structures of human zinc transporter ZnT7 reveal the mechanism of Zn2+ uptake into the Golgi apparatus

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+-bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a dimer via tight interactions in both the cytosolic and transmembrane (TM) domains of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to the TM metal transport pathway. These structures permit mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.

New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Design and development of novel potential inhibitors of the human USP21 enzyme using a pharmacophore-based virtual screening technique.

An overexpression and increase have been observed in the concentration and activity of the USP21 enzyme in many cancers, necessitating the need for the development of new inhibitor drugs against the same. The current study attempts to discover one such novel potential inhibitor of USP21 by the application of various bioinformatics techniques which include molecular modelling, pharmacophore mapping, and pharmacophore-based virtual screening, molecular docking, and ADMET prediction followed by molecular dynamics simulations. Following this inverted-funnel-like approach, we finally ended up with one ligand- ZINC02422616 which displays a very high binding affinity towards the USP21 domain. This ligand contains all the pharmacophoric features displayed by the compounds that are potential inhibitors of the USP21 domain. Moreover, it shows a favourable pharmacokinetic, pharmacodynamic, and ADMET profile along with strong hydrophobic interaction and hydrogen bonding with the domain. Simulation studies showed that the complex remains stable over time, with the bound protein displaying a more constrained motion in the conformational space compared to the unbound form. The ligand showed a highly favourable free energy landscape/surface, forming several energy minimas in contrast to the unbound domain in which most conformations occupied a relatively higher energy state. Moreover, the ligand also displayed a Kd of 422.8nM and a free energy of binding ΔG of -8.6kcal/mol, both of which indicate a very high affinity towards the target domain.This potential drug candidate can then be used as a viable treatment method for many types of cancers caused by USP21. his article is protected by copyright. All rights reserved.

Drug interactions between ALK inhibitors and warfarin with concurrent use of bucolome: a case report

BackgroundAlectinib, crizotinib, and ceritinib, are anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) that exhibit high protein binding, and their metabolism is associated with the cytochrome P450 (CYP) isoenzymes 2C9 or 3A4. The plasma protein binding rate of warfarin, which is used to prevent and treat venous thromboembolism, is also high. Warfarin is a racemate of S-warfarin and R-warfarin, which are metabolized by CYP2C9 and CYP3A4, respectively. Reports on the drug interactions between each of the above-mentioned ALK-TKIs and warfarin with concurrent use of bucolome are currently lacking.Case presentation.We report a case of a patient receiving warfarin and bucolome, whose international normalized ratio (INR) increased after sequential treatment with alectinib, crizotinib, and ceritinib. The patient was a 61-year-old man with a history of aortic valve regurgitation, who was receiving warfarin treatment following aortic valve replacement. Bucolome, which can enhance the effect of warfarin, was also used simultaneously. The patient was diagnosed with primary lung adenocarcinoma, and ALK rearrangement was detected during second-line chemotherapy. After progression of the disease with chemotherapy, sequential treatment with alectinib, crizotinib, and ceritinib was initiated. Pretreatment INR values were in the therapeutic range (target INR of 2–3) but increased to supratherapeutic levels each time after initiation of alectinib, crizotinib, or ceritinib treatment. Adjustment of warfarin dose or discontinuation of bucolome were necessary to maintain the therapeutic INR range. There were no serious bleeding events or substantial changes in dietary intake.Displacement of plasma protein binding or competitive inhibition of metabolism by alectinib, crizotinib, and ceritinib could increase the plasma concentration of the unbound form of warfarin, resulting in high INR values. In addition, alectinib, crizotinib, and ceritinib might cause displacement of bucolome from plasma proteins, followed by displacement of warfarin or inhibition of warfarin metabolism caused by the unbound form of bucolome.ConclusionsClose monitoring of INR and adjustment of warfarin dosage are needed during treatment with alectinib, crizotinib, or ceritinib in patients who receive warfarin with concurrent use of bucolome.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins

BackgroundIn spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host’s capsule.ResultsThe TP84_26 ‘capsule depolymerase’ (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC–MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion.ConclusionsThe novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

New Biosensor for Determination of Neuropilin-1 with Detection by Surface Plasmon Resonance Imaging.

Neuropilin-1 is transmembrane protein with soluble isoforms. It plays a pivotal role in both physiological and pathological processes. NRP-1 is involved in the immune response, formation of neuronal circuits, angiogenesis, survival and migration of cells. The specific SPRI biosensor for the determination of neuropilin-1 was constructed using mouse monoclonal antibody that captures unbound NRP-1 form body fluids. The biosensor exhibits linearity of the analytical signal between 0.01 and 2.5 ng/mL, average precision value 4.7% and recovery between 97% and 104%. The detection limit is 0.011 ng/mL, and the limit of quantification is 0.038 ng/mL. The biosensor was validated by parallel determination of NRP-1 in serum and saliva samples using the ELISA test, with good agreement of the results.

Abstract 3847: Target identification of a multi-pass transmembrane G protein coupled receptor using limited-proteolysis coupled mass spectrometry (LiP-MS)

Abstract In drug discovery, target identification (ID) after phenotypic screens is a resource-intensive endeavor aimed to understand compound’s mechanism of action. Target ID for membrane proteins is particularly challenging due to hurdles such as poor protein solubility, instability and low expression levels. Addressing these hurdles, recent proteomics-based strategies allow to analyze proteins in their native environment, and do not require compound modification or genetic manipulation of target cell lines. Limited proteolysis coupled to mass spectrometry (LiP-MS) is a peptide-centric strategy that exploits structural protein alterations and steric hindrances induced by drug to detect drug-protein interactions, estimate potency (EC50) and predict binding sites across the proteome. Our previous reports (AACR 2020 and 2021) showed the applicability of LiP-MS on cytosolic proteins such as kinases and phosphatases. Here, LiP-MS workflow was adapted for multi-pass membrane proteins which are often underrepresented in global, unbiased target ID approaches. For protein targets in the cytosol or intracellular organelles, LiP-MS deploys a dose-response (DR) analysis recorded by incubating cell lysates with a given compound. Here, to monitor proteins in plasma membrane, live cells were treated with the compound in a DR curve for a short period of time before lysis and subsequent LiP-MS analysis. Live cell treatment allows to achieve target ID by 1) locking the receptor in its native conformation to detect the structural difference between the drug-bound and unbound forms and; 2) detecting structural changes in downstream signaling cascades resulting from changes in protein-protein interactions, post-translational modifications or other mechanisms. We evaluated the performance of this workflow using a tool compound (IAX16840) targeting specific G-protein coupled receptors. Our unbiased LiP scoring identified atypical chemokine receptor 3 (ACKR3), the primary known compound target, among the top 3 hits. Additional 82 proteins were perturbed in drug-treated samples based on the LiP scores. Mapping the altered peptides on the GPCR signaling network showed enrichment of perturbations in ACKR3 downstream pathways, providing additional evidence of ACKR3 binding. Taken together, we demonstrate that the live cell LiP-MS is applicable for target ID of multi-pass membrane proteins. The approach also provides a system-level view of protein and pathway perturbations downstream of a signaling protein (e.g. GPCR), revealing possible mechanisms of its endogenous action. Further optimization of LiP-MS protocol is sought to accommodate different classes of receptors. Citation Format: Martin Soste, Dominique Kamber, Nadine Dobberstein, Mirjam Zimmermann, Aurelien Rizk, Yuehan Feng, Yaroslav Nikolaev, Nigel Beaton. Target identification of a multi-pass transmembrane G protein coupled receptor using limited-proteolysis coupled mass spectrometry (LiP-MS). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3847.

Binding mechanism of andrographolide with intramolecular antiparallel G-quadruplexes of therapeutic importance: an in-silico analysis

ABSTRACT G-quadruplexes are non-canonical DNA structures that emerged as important drug targets. Andrographolide is a plant-derived compound with various pharmacological properties. The binding of andrographolide with CREB1 G-quadruplex and Thrombin Binding Aptamer (TBA) was estimated using molecular docking, dynamics, and simulation. The data demonstrated the stabilising effect of the ligand on G-quadruplexes. Andrographolide binds to the CREB1 G-quadruplex by forming hydrogen bonds with the terminal residues and thymine and cytosine residues in the loop region with a binding energy of – 7.30 kcal/mol. At the same time, the TBA G-quadruplex structure formed hydrogen bonds with the residues in the quartet and loop region with binding energy – 6.61 kcal/mol. The Molecular Mechanics Poisson-Boltzmann Surface Area analysis revealed that the contribution from electrostatic energy is higher (−109.21 ± 51.40 KJ/mol) for CREB1 than for TBA (−41.49 ± 27.25 KJ/mol). Principle component analysis illustrated that the eigenvalues are much higher for the unbound forms than for the ligand-bound forms. These findings indicate that andrographolide could potentially target G-quadruplex forming DNA sequences in various oncogenes and can have multiple therapeutic applications.

Characterization of the Stereoselective Disposition of Bupropion and Its Metabolites in Rat Plasma and Brain.

Bupropion is an atypical antidepressant and smoking cessation aid; its use is associated with wide intersubject variability in efficacy and safety. Knowledge of the brain pharmacokinetics of bupropion and its pharmacologically active metabolites is considered important for understanding the cause-effect relationships driving this variability. Brain concentrations from rats administered a 10 mg/kg subcutaneous dose of racemic bupropion were analyzed using a stereoselective LC/MS-MS method. A 2 mg/kg dose of (S,S)-hydroxybupropion, which has comparable pharmacologic potency to bupropion, was administered to a separate group of rats. Plasma exposure and unbound concentrations in both matrices from companion equilibrium dialysis experiments were determined to assess potential carrier-mediated transport at the blood-brain barrier. Exposures to unbound forms of bupropion enantiomers were similar in plasma; this was also true in brain. This trend held for reductive diastereomer metabolite pairs in the two matrices. Unbound (R,R)-hydroxybupropion exposure was 1.5-fold higher than (S,S)-hydroxybupropion exposure in plasma and brain following bupropion administration. Unbound concentration ratios (Kp,uu) of a given molecular form decreased over time: between 4 and 6 h, these were < 1 for the two bupropion enantiomers, and they were ~ 1 for metabolites that formed. Administration of preformed (S,S)-hydroxybupropion also demonstrated a declining Kp,uu. The temporal shift in Kp,uu among the different molecular forms provides evidence regarding the operation of carrier-mediated transport and/or within-brain metabolism of bupropion, and, thereby, fresh insight regarding the causes of intersubject variability in the safety and efficacy of bupropion therapy.

Active-site electronic properties and conformational dynamics of pet-degrading enzymes.

Polyethylene terephthalate (PET) is one of the most widely used plastic polymers in the world. PET waste leads to the creation of micro- and nanosized plastic particles that have been linked to cell damage and inflammation in humans. Despite this worldwide environmental and health threat, accumulation of PET waste increases yearly because the main PET recycling method (thermomechanical) results in loss of its desired mechanical properties, which limits recycling. Reports of PET-degrading enzymes converting PET polymer into its molecular building blocks fuel circular economy concepts and a need to optimize these enzymes to achieve higher turnover rates for industrial applications. Here, we study PETase and MHETase from I. sakaiensis using molecular dynamics simulations with the Drude polarizable force field (Drude-2019). Parameters were developed to describe electronic features of PET polymer and its derived monomers, allowing us to study both enzymes in their bound and unbound forms. We found that the overall active site architecture in both PETase and MHETase is sensitive to the presence of substrate, suggesting a possible optimization target. Network analysis of enzyme dynamics suggests key residues that maintain binding site architecture. Moreover, dipole-dipole responses of PETase and MHETase binding site residues were also analyzed as a function of substrate binding. In addition, electronic properties of PET monomers were studied in multiple solvents with different dielectric properties to better understand the substrate polarization when bound to the enzyme. Electric field calculations allowed us to show how PETase and MHETase organize a electric fields that facilitate to catalysis. Overall, these results elucidate the interplay between electronic properties of PET-degrading enzymes and their conformational dynamics, opening new avenues for enzyme engineering efforts.

What does alphafold know about protein folding and peptide binding.

Machine learning approaches have recently revolutionized structural biology by predicting 3D structures of proteins at atomistic level from their sequence with high confidence and accuracy. The community is pushing the limits of interpretability and application of these algorithms beyond their original objective. Here, we present our studies on what deep neural network models for protein structure prediction such as AlphaFold can shed light on two fundamental biological processes: (1) protein folding and (2) protein-peptide binding. We first demonstrate our studies on deciphering the folding mechanism of protein G, L and their mutants, two pair of proteins with low sequence similarity that fold into the same topology. By using large-scale molecular dynamics simulations with advanced enhanced sampling methods and Markov state modeling analysis, we observed how the sequence difference impacts folding pathways and rate in unprecedented detail. Then, we show a novel fragment decomposition approach using AlphaFold to identify preferences for secondary structure element combinations that follow the order of events observed in the folding pathways. Standing upon the highly sensitive mapping of AlphaFold between the sequence and structure, we discovered how to directly use the model to identify high affinity peptide binders using a competitive binding assay. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form. The speed and robustness of the method will make it readily applicable in screening libraries of peptide sequences to prioritize for detailed experimental characterization.

Ranking Peptide Binders by Affinity with AlphaFold.

AlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein-peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.

Ranking Peptide Binders by Affinity with AlphaFold**

AbstractAlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein‐peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.

Native Mass Spectrometry of BRD4 Bromodomains Linked to a Long Disordered Region.

The contribution of disordered regions to protein function and structure is a relatively new field of study and of particular significance as their function has been implicated in some human diseases. Our objective was to analyze various deletion mutants of the bromodomain-containing protein 4 (BRD4) using native mass spectrometry to characterize the gas-phase behavior of the disordered region connected to the folded domain. A protein with a single bromodomain but no long disordered linker displayed a narrow charge distribution at low charge states, suggesting a compact structure. In contrast, proteins containing one or two bromodomains connected to a long disordered region exhibited multimodal charge distributions, suggesting the presence of compact and elongated conformers. In the presence of a pan-BET-bromodomain inhibitor, JQ1, the protein-JQ1 complex ions had relatively small numbers of positive charges, corresponding to compact conformers. In contrast, the ions with extremely high charge states did not form a complex with JQ1. This suggests that all of the JQ1-bound BRD4 proteins in the gas phase are in a compact conformation, including the linker region, while the unbound forms are considerably elongated. Although these are gas-phase phenomena, it is possible that the long disordered linker connected to the bromodomain causes the denaturation of the folded domain, which, in turn, affects its JQ1 recognition.

Impaired VEGF-A-Mediated Neurovascular Crosstalk Induced by SARS-CoV-2 Spike Protein: A Potential Hypothesis Explaining Long COVID-19 Symptoms and COVID-19 Vaccine Side Effects?

Long coronavirus disease-19 (COVID-19) is a newly discovered syndrome characterized by multiple organ manifestations that persist for weeks to months, following the recovery from acute disease. Occasionally, neurological and cardiovascular side effects mimicking long COVID-19 have been reported in recipients of COVID-19 vaccines. Hypothetically, the clinical similarity could be due to a shared pathogenic role of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike (S) protein produced by the virus or used for immunization. The S protein can bind to neuropilin (NRP)-1, which normally functions as a coreceptor for the vascular endothelial growth factor (VEGF)-A. By antagonizing the docking of VEGF-A to NRP-1, the S protein could disrupt physiological pathways involved in angiogenesis and nociception. One consequence could be the increase in unbound forms of VEGF-A that could bind to other receptors. SARS-CoV-2-infected individuals may exhibit increased plasma levels of VEGF-A during both acute illness and convalescence, which could be responsible for diffuse microvascular and neurological damage. A few studies suggest that serum VEGF-A may also be a potential biomarker for long COVID-19, whereas evidence for COVID-19 vaccines is lacking and merits further investigation.

Understanding protein diffusion on force-induced stretched DNA conformation.

DNA morphology is subjected to environmental conditions and is closely coupled with its function. For example, DNA experiences stretching forces during several biological processes, including transcription and genome transactions, that significantly alter its conformation from that of B-DNA. Indeed, a well-defined 1.5 times extended conformation of dsDNA, known as Σ-DNA, has been reported in DNA complexes with proteins such as Rad51 and RecA. A striking feature in Σ-DNA is that the nucleobases are partitioned into triplets of three locally stacked bases separated by an empty rise gap of Å. The functional role of such a DNA base triplet was hypothesized to be coupled with the ease of recognition of DNA bases by DNA-binding proteins (DBPs) and the physical origin of three letters (codon/anti-codon) in the genetic code. However, the underlying mechanism of base-triplet formation and the ease of DNA base-pair recognition by DBPs remain elusive. To investigate, here, we study the diffusion of a protein on a force-induced stretched DNA using coarse-grained molecular dynamics simulations. Upon pulling at the 3' end of DNA by constant forces, DNA exhibits a conformational transition from B-DNA to a ladder-like S-DNA conformation via Σ-DNA intermediate. The resulting stretched DNA conformations exhibit non-uniform base-pair clusters such as doublets, triplets, and quadruplets, of which triplets are energetically more stable than others. We find that protein favors the triplet formation compared to its unbound form while interacting non-specifically along DNA, and the relative population of it governs the ruggedness of the protein-DNA binding energy landscape and enhances the efficiency of DNA base recognition. Furthermore, we analyze the translocation mechanism of a DBP under different force regimes and underscore the significance of triplet formation in regulating the facilitated diffusion of protein on DNA. Our study, thus, provides a plausible framework for understanding the structure-function relationship between triplet formation and base recognition by a DBP and helps to understand gene regulation in complex regulatory processes.

Structural Landscape of nsp Coding Genomic Regions of SARS-CoV-2-ssRNA Genome: A Structural Genomics Approach Toward Identification of Druggable Genome, Ligand-Binding Pockets, and Structure-Based Druggability.

SARS-CoV-2 has a single-stranded RNA genome (+ssRNA), and synthesizes structural and non-structural proteins (nsps). All 16 nsp are synthesized from the ORF1a, and ORF1b regions associated with different life cycle preprocesses, including replication. The regions of ORF1a synthesizes nsp1 to 11, and ORF1b synthesizes nsp12 to 16. In this paper, we have predicted the secondary structure conformations, entropy & mountain plots, RNA secondary structure in a linear fashion, and 3D structureof nsp coding genes of the SARS-CoV-2 genome. We have also analyzed the A, T, G, C, A+T, and G+C contents, GC-profiling of these genes, showing the range of the GC content from 34.23 to 48.52%. We have observed that the GC-profile value of the nsp coding genomic regions was less (about 0.375) compared to the whole genome (about 0.38). Additionally, druggable pockets were identified from the secondary structure-guided 3D structural conformations. For secondary structure generation of all the nsp coding genes (nsp 1-16), we used a recent algorithm-based tool (deep learning-based) along with the conventional algorithms (centroid and MFE-based) to develop secondary structural conformations, and we found stem-loop, multi-branch loop, pseudoknot, and the bulge structural components, etc. The 3D model shows bound and unbound forms, branched structures, duplex structures, three-way junctions, four-way junctions, etc. Finally, we identified binding pockets ofnsp coding genes which will help as a fundamental resource for future researchers to develop RNA-targeted therapeutics using the druggable genome.

Pathophysiology of intraocular haemorrhage: Role of iron in cell death

AbstractRetinal haemorrhages are present in several retinal pathologies, such as exudative AMD, diabetic retinopathy, or myopic degeneration. As a consequence of erythrocytes lysis following haemorrhage, haemoglobin (Hb) is released into the extracellular space, and heme is degraded into iron, which becomes under a free (unbound) form. While iron is essential for retinal functions, labile free iron enhances Fenton reaction and therefore leads to oxidative stress. Cones are the most sensitive retinal cells to iron excess and local accumulation of iron rapidly induces photoreceptors and retinal pigment epithelium cells death. A local retinal homeostasis of iron exists to equilibrate iron level in retinal cells, but a deficiency of iron‐related proteins causes iron accumulation and retinal degeneration. Heme‐binding proteins, hemopexin and haptoglobin, and their receptors, as well as heme transporters were also found expressed in retinal cells suggesting their roles in retinal iron regulation. As therapeutic strategy, chemical chelators used to control iron accumulation in systemic iron overload diseases, have demonstrated efficiency to protects the retina in various models of retinal degenerations. Our team uses transferrin (TF), the iron transporter protein, to reduce iron overload and therefore preserve visual function. Control of iron impairment in retinal tissues may be a therapeutic strategy during haemorrhages.

Structures of the CcmABCD heme release complex at multiple states

Cytochromes c use heme as a cofactor to carry electrons in respiration and photosynthesis. The cytochrome c maturation system I, consisting of eight membrane proteins (CcmABCDEFGH), results in the attachment of heme to cysteine residues of cytochrome c proteins. Since all c-type cytochromes are periplasmic, heme is first transported to a periplasmic heme chaperone, CcmE. A large membrane complex, CcmABCD has been proposed to carry out this transport and linkage to CcmE, yet the structural basis and mechanisms underlying the process are unknown. We describe high resolution cryo-EM structures of CcmABCD in an unbound form, in complex with inhibitor AMP-PNP, and in complex with ATP and heme. We locate the ATP-binding site in CcmA and the heme-binding site in CcmC. Based on our structures combined with functional studies, we propose a hypothetic model of heme trafficking, heme transfer to CcmE, and ATP-dependent release of holoCcmE from CcmABCD. CcmABCD represents an ABC transporter complex using the energy of ATP hydrolysis for the transfer of heme from one binding partner (CcmC) to another (CcmE).

A dynamical view of protein-protein complexes: Studies by molecular dynamics simulations.

Protein-protein interactions are at the basis of many protein functions, and the knowledge of 3D structures of protein-protein complexes provides structural, mechanical and dynamical pieces of information essential to understand these functions. Protein-protein interfaces can be seen as stable, organized regions where residues from different partners form non-covalent interactions that are responsible for interaction specificity and strength. They are commonly described as a peripheral region, whose role is to protect the core region that concentrates the most contributing interactions, from the solvent. To get insights into the dynamics of protein-protein complexes, we carried out all-atom molecular dynamics simulations in explicit solvent on eight different protein-protein complexes of different functional class and interface size by taking into account the bound and unbound forms. On the one hand, we characterized structural changes upon binding of the proteins, and on the other hand we extensively analyzed the interfaces and the structural waters involved in the binding. Based on our analysis, in 6 cases out of 8, the interfaces rearranged during the simulation time, in stable and long-lived substates with alternative residue-residue contacts. These rearrangements are not restricted to side-chain fluctuations in the periphery but also affect the core interface. Finally, the analysis of the waters at the interface and involved in the binding pointed out the importance to take into account their role in the estimation of the interaction strength.