Papers by Alexander Yakimov
Journal of Biomedicine and Biotechnology, 2012
Maintenance of the cellular redox balance has vital importance for correcting organism functionin... more Maintenance of the cellular redox balance has vital importance for correcting organism functioning. Methionine sulfoxide reductases (Msrs) are among the key members of the cellular antioxidant defence system. To work properly, methionine sulfoxide reductases need to be reduced by their biological partner, thioredoxin (Trx). This process, according to the available kinetic data, represents the slowest step in the Msrs catalytic cycle. In the present paper, we investigated structural aspects of the intermolecular complex formation between mammalian MsrB1 and Trx. NMR spectroscopy and biocomputing were the two mostly used through the research approaches. The formation of NMR detectable MsrB1/Trx complex was monitored and studied in attempt to understand MsrB1 reduction mechanism. Using NMR data, molecular mechanics, protein docking, and molecular dynamics simulations, it was found that intermediate MsrB1/Trx complex is stabilized by interprotein β-layer. The complex formation accompanied by distortion of disulfide bond within MsrB1 facilitates the reduction of oxidized MsrB1 as it is evidenced by the obtained data.
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes ... more Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes of redox reactions in central metabolic pathways. NAD is also used to generate second messengers (such as cyclic ADP-ribose) and serves as substrate for protein modifications (including ADP-ribosylation and protein deacetylation). The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells regulate their NAD supply through biosynthesis using various precursors: nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are then adenylylated to the dinucleotides NAD or NAAD, respectively. Here, we have developed NMR-based experimental approach to identify NAD and its intermediates in culture of human cells. Using this method we have detected and quantified NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in the standard culture medium containing Nam as the only NAD precursor. When cells were grown in the presence of NR and NAR, we have additionally identified intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD). Also, we have characterized the potential of different extracellular NAD precursors to maintain the synthesis of intracellular NAD.
Journal of Biological Chemistry
Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) i... more Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.
Biochemical and Biophysical Research Communications
RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA... more RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA repair, natural transformation and SOS-response activation. RecA forms nucleoprotein filaments on single-stranded DNA with a highly conserved architecture that is also shared by eukaryotic recombinases. One of the key features of these filaments is the ability to switch between stretched and compressed conformations in response to ATP binding and hydrolysis. However, the functional role of such conformational changes is not fully understood. Structural data revealed that in the absence of ATP RecA binds DNA with the stoichiometry of 5 nucleotides per one monomer, while in the presence of ATP the binding stoichiometry is 3:1. Such differences suggest incompatibility of the active and inactive conformations, yet dynamic single-molecule studies demonstrated that ATP and apo conformations can be directly interconvertible. In the present work we use a single-molecule approach to address the features of inactive RecA nucleoprotein filaments formed de novo in the absence of nucleotide cofactors. We show that compressed RecA-DNA filaments can exist with both 5:1 and 3:1 binding stoichiometry which is determined by conditions of the filament assembly. However, only a 3:1 stoichiometry allows direct interconvertibility with the active ATP-bound conformation.
FEBS Letters, 2020
The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through ass... more The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single‐stranded DNA in the presence of ATP. Large‐scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single‐molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer–monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA‐mediated processes of HR.
RecA protein mediates homologous recombination repair in bacteria through assembly of long helica... more RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.
International Journal of Molecular Sciences, 2021
Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide a... more Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cel...
International Journal of Molecular Sciences, 2020
Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of w... more Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational sw...
Nucleic Acids Research, 2019
A large variety of short biologically active peptides possesses antioxidant, antibacterial, antit... more A large variety of short biologically active peptides possesses antioxidant, antibacterial, antitumour, anti-ageing and anti-inflammatory activity, involved in the regulation of neuro-immuno-endocrine system functions, cell apoptosis, proliferation and differentiation. Therefore, the mechanisms of their biological activity are attracting increasing attention not only in modern molecular biology, biochemistry and biophysics, but also in pharmacology and medicine. In this work, we systematically analysed the ability of dipeptides (all possible combinations of the 20 standard amino acids) to bind all possible combinations of tetra-nucleotides in the central part of dsDNA in the classic B-form using molecular docking and molecular dynamics. The vast majority of the dipeptides were found to be unable to bind dsDNA. However, we were able to identify 57 low-energy dipeptide complexes with peptide-dsDNA possessing high selectivity for DNA binding. The analysis of the dsDNA complexes with di...
This manuscript describes the chemical synthesis of a compound similar to fluorene and Congo red,... more This manuscript describes the chemical synthesis of a compound similar to fluorene and Congo red, including characterization of its spectral properties. It was shown that the dye, during interaction with amyloid-like fibrils of several proteins (lysozyme, insulin, and beta-2-microglobulin), has the ability to change fluorescent spectrum. In contrast, monomeric forms of these proteins did not induce significant spectral changes.
Acta Naturae, 2016
РЕФЕРАТ Наиболее часто встречающимся элементом вторичной структуры во многих глобулярных водораст... more РЕФЕРАТ Наиболее часто встречающимся элементом вторичной структуры во многих глобулярных водорастворимых белках и пептидах являются α-спирали. Повышенная конформационная стабильность α-спиралей-одна из главных причин высокой термостабильности белков термофильных бактерий. Кроме того, α-спирали часто участвуют во взаимодействиях белков не только с другими белками, но и с нуклеиновыми кислотами, а также с липидами клеточных мембран. Именно поэтому конструирование высокостабильных α-спиральных пептидов, используемых в качестве высокоактивных и высокоспецифичных ингибиторов межбелковых и других взаимодействий, в последнее время находит все больше практических применений в медицине. Для улучшения конформационной стабильности α-спиральных пептидов и термостабильных белков в последнее время разработано несколько подходов, которые мы обсудим в этом обзоре. Кроме того, будут рассмотрены методы улучшения прохождения пептидов и белков через клеточные мембраны и устойчивости к действию внутриклеточных протеаз. Особое внимание уделено методу SEQOPT (http://mml.spbstu.ru/services/seqopt/), который используется для конструирования конформационно стабильных коротких α-спиралей. КЛЮЧЕВЫЕ СЛОВА α-спираль, конформационная стабильность, проницаемость мембран, сопротивляемость внутриклеточному протеолизу, факторы термостабильности. СОКРАЩЕНИЯ SEQOPT-метод глобальной оптимизации аминокислотных последовательностей α-спиралей; МД-метод молекулярной динамики; HC (alpha-helix content)-содержание (%) α-спиральной конформации в белке; PDB-база данных пространственных структур белков; AGADIR, ALB, HELIX-статистико-механические модели, описывающие конформационные переходы α-спираль-клубок в коротких мономерных пептидах; КД-спектроскопия кругового дихроизма.
Cell and Tissue Biology, 2018
Nicotinamide adenine dinucleotide (NAD) plays a key role in the vital metabolic and regulatory pr... more Nicotinamide adenine dinucleotide (NAD) plays a key role in the vital metabolic and regulatory processes in mammals. Disturbance of the NAD level regulation is associated with the development of such serious diseases as pellagra, neurodegenerative and cardiovascular disorders, diabetes, cancer and others. This paper presents an experimental approach that allows to determine the amount of NAD + in mouse tissues using NMR spectroscopy, as well as the level of NAD +-dependent deacetylation of proteins in the cytosol and mitochondria.
International Journal of Molecular Sciences, 2018
Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzyme... more Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method,...
Cell and Tissue Biology, 2016
TIP49a and TIP49b, highly conserved proteins belonging to the AAA + superfamily of DNA dependent ... more TIP49a and TIP49b, highly conserved proteins belonging to the AAA + superfamily of DNA dependent ATPases, participate in numerous cell processes, such as chromatin remodeling, regulation of gene transcription and mitotic cell division, maintenance of genome stability, and snoRNP biogenesis, as well as in the formation of active DNA-telomerase complexes. It has been shown that they are involved in com plex networks of protein-protein interactions and, in spite of their structural similarity, sometimes perform opposite functions. Although these proteins exhibit a wide range of activities, the mechanisms of their actions are still poorly understood. In this work, ring shaped heterohexameric TIP49a/b complexes containing in their central channel short fragments of double stranded DNA (dsDNA, 20 base pairs of different GC com position) were obtained for the first time using the molecular docking technique, while methods of molecular dynamics in a periodic water box were applied to investigate the conformational dynamics of these proteins and the mechanisms of their helicase activity. It was found that (1) interaction of a DNA helix with positively charged protein loops inside the central channel of the ring shaped hexameric complex caused unwinding of the helix; (2) the unwinding occurred only inside the hexameric ring, whereas the tails of the helix, which lie outside, retained the initial classical B form conformation throughout the 50 ns of molecular dynamics; and (3) the presence of ATP in TIP49a/b complexes affected the dynamics and the final structure of dsDNA, causing partial breaking of complementary bonds in GC poor DNA sequences.
Nucleic Acids Research, 2017
The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA f... more The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class ␣-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein ␣-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.
Nature chemical biology, 2017
Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms ... more Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering with translation elongation. Structural analysis of the ribosome-KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs...
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Papers by Alexander Yakimov