139 research outputs found
Halogen bonding at the ATP binding site of protein kinases: Preferred geometry and topology of ligand binding
ABSTRACT Halogenated ligands have been widely developed as potent, and frequently selective, inhibitors of protein kinases (PK). Herein, all structures of protein kinases complexed with a halogenated ligand, identified in the PDB, were analyzed in the context of eventual contribution of halogen bonding to protein-ligand interactions. Global inspection shows that two carbonyl groups of residues located in the hinge region are the most abundant halogen bond acceptors. In contrast to solution data, well-defined water molecules, located at sites conserved across most PK structures, are also involved in halogen bonding. Analysis of cumulative distributions of halogen-acceptor distances shows that structures displaying short contacts involving a halogen atom are overpopulated, contributing together to clearly defined maxima of 2.82, 2.91 and 2.94 Å for chlorine, bromine and iodine, respectively. The angular preference of a halogen bond favors ideal topology (180°, 120°) for iodine. For bromine the distribution is much more dispersed, and no such preference was found for chlorine
Halogen bonds involved in binding of halogenated ligands by protein kinases.
Analysis of 664 known structures of protein kinase complexes with halogenated ligands revealed 424 short contacts between a halogen atom and a potential protein X-bond acceptor, the topology and geometry of which were analyzed according to the type of a halogen atom (X = Cl, Br, I) and a putative protein X-bond acceptor. Among 236 identified halogen bonds, the most represented ones are directed to backbone carbonyls of the hinge region and may replace the pattern of ATP-like hydrogen bonds. Some halogen-Ï€ interactions with either aromatic residues or peptide bonds, that accompany the interaction with the hinge region, may possibly enhance ligand selectivity. Interestingly, many of these halogen-Ï€ interactions are bifurcated. Geometrical preferences identify iodine as the strongest X-bond donor, less so bromine, while virtually no such preferences were observed for chlorine; and a backbone carbonyl as the strongest X-bond acceptor. The presence of a halogen atom in a ligand additionally affects the properties of proximal hydrogen bonds, which according to geometrical parameters get strengthened, when a nitrogen of a halogenated ligand acts as the hydrogen bond donor
Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions.
The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called ‘thymine-less death’ route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours,however,theinitialsteady-statewaspreservedonlyforanhour,untiltheATPconcentration dropped by a half, which was monitored independently via 31P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast
1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase
1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA
Arteriovenous oscillations of the redox potential: Is the redox state influencing blood flow?
OBJECTIVE
Studies on the regulation of human blood flow revealed several modes of oscillations with frequencies ranging from 0.005 to 1 Hz. Several mechanisms were proposed that might influence these oscillations, such as the activity of vascular endothelium, the neurogenic activity of vessel wall, the intrinsic activity of vascular smooth muscle, respiration, and heartbeat. These studies relied typically on non-invasive techniques, for example, laser Doppler flowmetry. Oscillations of biochemical markers were rarely coupled to blood flow.
METHODS
The redox potential difference between the artery and the vein was measured by platinum electrodes placed in the parallel homonymous femoral artery and the femoral vein of ventilated anesthetized pigs.
RESULTS
Continuous measurement at 5 Hz sampling rate using a digital nanovoltmeter revealed fluctuating signals with three basic modes of oscillations: ∼ 1, ∼ 0.1 and ∼ 0.01 Hz. These signals clearly overlap with reported modes of oscillations in blood flow, suggesting coupling of the redox potential and blood flow.
DISCUSSION
The amplitude of the oscillations associated with heart action was significantly smaller than for the other two modes, despite the fact that heart action has the greatest influence on blood flow. This finding suggests that redox potential in blood might be not a derivative but either a mediator or an effector of the blood flow control system
Similar but Not Identical—Binding Properties of LSU (Response to Low Sulfur) Proteins From Arabidopsis thaliana
Members of the plant-specific LSU (RESPONSE TO LOW SULFUR) family are strongly induced during sulfur starvation. The molecular functions of these proteins are unknown; however, they were identified as important stress-related hubs in several studies. In Arabidopsis thaliana, there are four members of the LSU family (LSU1–4). These proteins are small (approximately 100 amino acids), with coiled-coil structures. In this work, we
investigated interactions between different monomers of LSU1–4. Differences in homo and heterodimer formation were observed. Our structural models of LSU1–4 homo- and
heterodimers were in agreement with our experimental observations and may help understand their binding properties. LSU proteins are involved in multiple protein–protein interactions, with the literature suggesting they can integrate abiotic and biotic stress responses. Previously, LSU partners were identified using the yeast two hybrid approach, therefore we sought to determine proteins co-purifying with LSU family members using protein extracts isolated from plants ectopically expressing TAP-tagged LSU1–4 constructs. These experiments revealed 46 new candidates for LSU partners. We tested four of them (and two other proteins, CAT2 and NBR1) for interaction with LSU1–4 by other methods. Binding of all six proteins with LSU1–4 was confirmed by Bimolecular Fluorescence Complementation, while only three of them were interacting with LSUs in yeast-two-hybrid. Additionally, we conducted network analysis of LSU interactome and revealed novel clues for the possible cellular function of these proteins
Rational drug-design approach supported with thermodynamic studies — a peptide leader for the efficient bi-substrate inhibitor of protein kinase CK2
Numerous inhibitors of protein kinases act on the basis of competition, targeting the ATP binding site. In this work, we present a procedure of rational design of a bi-substrate inhibitor, complemented with biophysical assays. The inhibitors of this type are commonly engineered by combining ligands carrying an ATP-like part with a peptide or peptide-mimicking fragment that determines specificity. Approach presented in this paper led to generation of a specific system for independent screening for efficient ligands and peptides, by means of thermodynamic measurements, that assessed the ability of the identified ligand and peptide to combine into a bi-substrate inhibitor. The catalytic subunit of human protein kinase CK2 was used as the model target. Peptide sequence was optimized using peptide libraries [KGDE]-[DE]-[ST]-[DE]3–4-NH2, originated from the consensus CK2 sequence. We identified KESEEE-NH2 peptide as the most promising one, whose binding affinity is substantially higher than that of the reference RRRDDDSDDD peptide. We assessed its potency to form an efficient bisubstrate
inhibitor using tetrabromobenzotriazole (TBBt) as the model ATP-competitive inhibitor. The formation of ternary complex was monitored using Differential Scanning Fluorimetry (DSF), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC)
Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel β-globin mutation
Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by
erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as
hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated
with β-thalassemia.
Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential
pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling.
Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G,
p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a
12-nucleotide deletion in the β-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence
of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the
disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four
PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic.
Identification of causative mutations should improve the diagnosis and management of HX and provide a new
insight into the molecular basis of this complex red blood cell abnormality
Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase
HIV-1 reverse transcriptase (HIV-1 RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical crosslinking method together with MD simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly-rA/dT tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly-rA/dT tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the polyrA/dT segment and help advance our understanding of the mechanisms in viral RNA reverse transcription
Native Structure-Based Peptides as Potential Protein–Protein Interaction Inhibitors of SARS-CoV-2 Spike Protein and Human ACE2 Receptor
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive-strand RNA virus that causes severe respiratory syndrome in humans, which is now referred to as coronavirus disease 2019 (COVID-19). Since December 2019, the new pathogen has rapidly spread globally, with over 65 million cases reported to the beginning of December 2020, including over 1.5 million deaths. Unfortunately, currently, there is no specific and effective treatment for COVID-19. As SARS-CoV-2 relies on its spike proteins (S) to bind to a host cell-surface receptor angiotensin-converting enzyme-2(ACE2), and this interaction is proved to be responsible for entering a virus into host cells, it makes an ideal target for antiviral drug development. In this work, we design three very short peptides based on the ACE2 sequence/structure fragments, which may effectively bind to the receptor-binding domain (RBD) of S protein and may, in turn, disrupt the important virus-host protein–protein interactions, blocking early steps of SARS-CoV-2 infection. Two of our peptides bind to virus protein with affinity in nanomolar range, and as very short peptides have great potential for drug development
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