Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic act... more Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We...
The synthetic function-spacer-lipid (FSL) amphiphile biotin-CMG-DOPE is widely used for delicate ... more The synthetic function-spacer-lipid (FSL) amphiphile biotin-CMG-DOPE is widely used for delicate ligation of living cells with biotin residues under physiological conditions. Since this molecule has an "apolar-polar-hydrophobic" gemini structure, the supramolecular organization is expected to differ significantly from the classical micelle. Its organization is investigated with experimental methods and molecular dynamics simulations (MDS). Although the linear length of a single biotin-CMG-DOPE molecule is 9.5 nm, the size of the dominant supramer globule is only 14.6 nm. Investigations found that while the DOPE tails form a hydrophobic core, the polar CMG spacer folds back upon itself and predominantly places the biotin reside inside the globule or planar layer. MDS demonstrates that < 10 % of biotin residues on the highly water dispersible globules and only 1 % of biotin residues in layer coatings are in an linear conformation and exposing biotin into the aqueous medium. This explains why in biotin-CMG-DOPE apolar biotin residues both in water dispersible globules and coatings on solid surfaces are still capable of interacting with streptavidin.
Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excess... more Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive. How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions. The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.
Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot... more Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot of structural data is available for the extracellular and cytoplasmic domains of TLRs, and a model of the dimeric full-length TLR3 receptor in the active state was build, the conformation of the transmembrane (TM) domain and juxtamembrane regions in TLR dimers is still unclear. In the present work, we study the transmembrane and juxtamembrane parts of human TLR4 receptor using solution NMR spectroscopy in a variety of membrane mimetics, including phospholipid bicelles. We show that the juxtamembrane hydrophobic region of TLR4 includes a part of long TM α-helix. We report the dimerization interface of the TM domain and claim that long TM domains with transmembrane charged aminoacids is a common feature of human toll-like receptors. This fact is analyzed from the viewpoint of protein activation mechanism, and a model of full-length TLR4 receptor in the dimeric state has been proposed.
Journal of biomolecular structure & dynamics, Jan 6, 2017
Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- a... more Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by (1)H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the s...
Lipid membranes are extremely stable envelopes allowing cells to survive in various environments ... more Lipid membranes are extremely stable envelopes allowing cells to survive in various environments and to maintain desired internal composition. Membrane permeation through formation of transversal pores requires substantial external stress. Practically, pores are usually formed by application of lateral tension or transmembrane voltage. Using the same approach as was used for obtaining continuous trajectory of pore formation in the stress-less membrane in the previous article, we now consider the process of pore formation under the external stress. The waiting time to pore formation proved a nonmonotonous function of the lateral tension, dropping from infinity at zero tension to a minimum at the tension of several millinewtons per meter. Transmembrane voltage, on the contrary, caused the waiting time to decrease monotonously. Analysis of pore formation trajectories for several lipid species with different spontaneous curvatures and elastic moduli under various external conditions provided instrumental insights into the mechanisms underlying some experimentally observed phenomena.
Lipid membranes serve as effective barriers allowing cells to maintain internal composition diffe... more Lipid membranes serve as effective barriers allowing cells to maintain internal composition differing from that of extracellular medium. Membrane permeation, both natural and artificial, can take place via appearance of transversal pores. The rearrangements of lipids leading to pore formation in the intact membrane are not yet understood in details. We applied continuum elasticity theory to obtain continuous trajectory of pore formation and closure, and analyzed molecular dynamics trajectories of pre-formed pore reseal. We hypothesized that a transversal pore is preceded by a hydrophobic defect: intermediate structure spanning through the membrane, the side walls of which are partially aligned by lipid tails. This prediction was confirmed by our molecular dynamics simulations. Conversion of the hydrophobic defect into the hydrophilic pore required surmounting some energy barrier. A metastable state was found for the hydrophilic pore at the radius of a few nanometers. The dependence of the energy on radius was approximately quadratic for hydrophobic defect and small hydrophilic pore, while for large radii it depended on the radius linearly. The pore energy related to its perimeter, line tension, thus depends of the pore radius. Calculated values of the line tension for large pores were in quantitative agreement with available experimental data.
The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kin... more The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein-lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated act...
Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic p... more Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N-and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O-and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed "iris-like" symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity.
Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family tran... more Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.
Abstract: Membrane and membrane-active peptides and proteins play a crucial role in numerous cell... more Abstract: Membrane and membrane-active peptides and proteins play a crucial role in numerous cell processes, such as signaling, ion conductance, fusion, and others. Many of them act as highly specific and efficient drugs or drug targets, and, therefore, attract ...
Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large-scale structural rearrangement... more Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large-scale structural rearrangements during spliceosome assembly, activation and catalysis. This is exemplified by the highly conserved U6 snRNA which can exist in isolation in the U6 small nuclear ribonucleoprotein (snRNP); basepaired with U4 in the U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and spliceosomal B complex; or basepaired with the pre-mRNA intron and the U2 snRNA in the catalytic spliceosome. Through genetic evidence and structure probing (1, 2), it was previously proposed that the U4/U6 di-snRNA consisted of a three-helical junction core domain flanked by single stranded regions. Using single-molecule Förster resonance energy transfer (smFRET), we discovered a dynamic equilibrium between RNA conformations formed by the flanking single stranded regions. Our smFRET data is consistent with the presence of two mutually exclusive structures: (1) a U4/U6 di-snRNA containing both basepaired U4/U6 and the U6 telestem and (2) a U4/U6 di-snRNA with extended basepairing located near U4/U6 stem I. These data show that the U6 telestem persists in the U4/U6 di-snRNA and that its formation can be regulated by novel basepairing interactions with U4. This suggests conservation of mechanism between the yeast and human U4/ U6 di-snRNAs. Finally, we speculate that telestem formation can be dynamically regulated by spliceosomal proteins to aid in tri-snRNP disassembly and spliceosome activation_rationalizing previous observations concerning the mechanism of the U4/U6 RNA helicase Brr2.
Specific interactions between transmembrane α-helices, to a large extent, determine the biologica... more Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions betwee...
Journal of bioinformatics and computational biology, 2007
Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc... more Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc2a from the latarcin family. The peptides were studied via long-term molecular dynamics (MD) simulations in different membrane environments (detergent micelles, mixed lipid bilayers mimiking eukaryotic and bacterial membranes). The calculated structure of Ltc2a in sodium dodecyl sulfate micelle agrees well with the data obtained by (1)H-NMR spectroscopy. This validates the applied modeling approach. The binding mode of MLPs is governed by several factors: (i) the membrane surface curvature; (ii) the conformational plasticity and hydrophobic organization of the peptide, which depend on the arrangement of charged, non-polar and helix-breaking residues in the amino acid sequence. In contrast to Ltc1, insertion of Ltc2a into model membranes induces significant changes in dynamic behavior of lipids in the contact region. Such a prominent membrane destabilization correlates with high membrane-...
Biochemical and biophysical research communications, Jan 7, 2014
Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammator... more Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.
Advances in protein chemistry and structural biology, 2011
This review surveys recent achievements of molecular computer modeling in understanding spatial s... more This review surveys recent achievements of molecular computer modeling in understanding spatial structure, dynamics, and mechanisms of functioning of transmembrane α-helical dimers in membranes. The factors driving self-association of hydrophobic helices in the membrane milieu are considered with examples of their applications to biologically relevant problems. The emphasis is made on the recent results, which help to understand important aspects of structure-function relations for these systems and their biological activity. Limitations and shortcomings of the methods, along with their perspectives in design of new membrane active agents, are discussed.
The hypothesis that local conformational differences of snake venom cardiotoxins (cytotoxins, CTs... more The hypothesis that local conformational differences of snake venom cardiotoxins (cytotoxins, CTs) may play a significant role in their interaction with membrane was tested by molecular modeling of the behavior of the CT A5 from the venom of Naja atra in water and at the water-membrane interface. Two models of the CT A5 spatial structure are known: the first was obtained by X-ray analysis and the second, by NMR studies in solution. A molecular dynamics (MD) analysis demonstrated that loop II of the toxin has a fixed omega-like shape in water, which does not depend on its initial structure. Interaction of the experimentally derived (X-ray and NMR) conformations and MD-simulated conformations of CT A5 with the lipid bilayer was studied by the Monte Carlo method using the previously developed model of the implicit membrane. The following was found: (1) Unlike the previously studied CT2 from the venom of cobra Naja oxiana, CT A5 has only loops I and II bound to the membrane, with the in...
unsupported bilayers. Recent experiments show that zwitterionic lipid bilayers supported on parti... more unsupported bilayers. Recent experiments show that zwitterionic lipid bilayers supported on partially charged, nanoporous silica wafers also do not interact with silica directly. However, in such cases the bilayer is separated from the silica substrate by a relative thicker (~1.5 nm) layer of solvent. We report here results from our MD simulation that reconcile these two seemingly disparate observations. We find that LJ nano-substrates whose porosity and surface hydroxyl density match those of nanoporous silica wafers do not increase the substrate-bilayer distance. Instead, the introduction of partial charge on the LJ substrate increases the substrate-bilayer distance to 1.5 nm. The negative charge on the substrate attracts hydrated ions, creating an electric double layer, which is a sufficient condition to increase the substrate-bilayer distance. While the properties of the bilayer supported on a charged substrate are also different from those of unsupported bilayers, the extent of the perturbation is not as large compared to that induced by uncharged hydroxylated substrates.
Ela®n, a speci®c inhibitor of elastase, is thought to play a regulatory role in in¯ammation. An N... more Ela®n, a speci®c inhibitor of elastase, is thought to play a regulatory role in in¯ammation. An NMR-derived solution structure of recombinant ela®n has been reported [Francart et al. (1997) J Mol Biol 268:666 ], although the conformation of its¯exible N-terminal part is not established. There is experimental evidence that the N terminus (residues 1±15) of ela®n interacts with the cell membrane. To explore the conformational preferences of residues in this region, we have performed Monte Carlo simulations of the peptide in water, in cyclohexane, and in a model membrane. Additionally, 3.7-ns molecular dynamics with explicit water was carried out. The main results were that the hydrophobic environment stabilizes an a helix in the region 6±11, the peptide is unordered in water, and it is attached to the membrane via the amphiphilic a-helix 6±11, which inserts with its N terminus forming an angle of about 60°to the membrane plane. We therefore assume that in nonpolar media the N-terminal part of ela®n forms a short a helix which might act as a membrane anchor.
Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic act... more Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We...
The synthetic function-spacer-lipid (FSL) amphiphile biotin-CMG-DOPE is widely used for delicate ... more The synthetic function-spacer-lipid (FSL) amphiphile biotin-CMG-DOPE is widely used for delicate ligation of living cells with biotin residues under physiological conditions. Since this molecule has an "apolar-polar-hydrophobic" gemini structure, the supramolecular organization is expected to differ significantly from the classical micelle. Its organization is investigated with experimental methods and molecular dynamics simulations (MDS). Although the linear length of a single biotin-CMG-DOPE molecule is 9.5 nm, the size of the dominant supramer globule is only 14.6 nm. Investigations found that while the DOPE tails form a hydrophobic core, the polar CMG spacer folds back upon itself and predominantly places the biotin reside inside the globule or planar layer. MDS demonstrates that < 10 % of biotin residues on the highly water dispersible globules and only 1 % of biotin residues in layer coatings are in an linear conformation and exposing biotin into the aqueous medium. This explains why in biotin-CMG-DOPE apolar biotin residues both in water dispersible globules and coatings on solid surfaces are still capable of interacting with streptavidin.
Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excess... more Spider venom is a rich source of antibacterial peptides, whose hemolytic activity is often excessive. How to get rid of it? Using latarcins from Lachesana tarabaevi and oxyopinin Oxt 4a from Oxyopes takobius spider venoms we performed coarse-grained molecular dynamics simulations of these peptides in the presence of lipid bilayers, mimicking erythrocyte membranes. This identified hemolytically active fragments within Oxt 4a and latarcins. Then, we synthesized five 20-residue peptides, containing different parts of the Oxt 4a and latarcin-1 sequence, carrying mutations within the identified regions. The antibacterial and hemolytic tests suggested that the three of the synthesized peptides demonstrated substantial decrease in hemolytic activity, retaining, or even exceeding antibacterial potential of the parent peptides.
Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot... more Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot of structural data is available for the extracellular and cytoplasmic domains of TLRs, and a model of the dimeric full-length TLR3 receptor in the active state was build, the conformation of the transmembrane (TM) domain and juxtamembrane regions in TLR dimers is still unclear. In the present work, we study the transmembrane and juxtamembrane parts of human TLR4 receptor using solution NMR spectroscopy in a variety of membrane mimetics, including phospholipid bicelles. We show that the juxtamembrane hydrophobic region of TLR4 includes a part of long TM α-helix. We report the dimerization interface of the TM domain and claim that long TM domains with transmembrane charged aminoacids is a common feature of human toll-like receptors. This fact is analyzed from the viewpoint of protein activation mechanism, and a model of full-length TLR4 receptor in the dimeric state has been proposed.
Journal of biomolecular structure & dynamics, Jan 6, 2017
Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- a... more Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from N.oxiana venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by (1)H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the s...
Lipid membranes are extremely stable envelopes allowing cells to survive in various environments ... more Lipid membranes are extremely stable envelopes allowing cells to survive in various environments and to maintain desired internal composition. Membrane permeation through formation of transversal pores requires substantial external stress. Practically, pores are usually formed by application of lateral tension or transmembrane voltage. Using the same approach as was used for obtaining continuous trajectory of pore formation in the stress-less membrane in the previous article, we now consider the process of pore formation under the external stress. The waiting time to pore formation proved a nonmonotonous function of the lateral tension, dropping from infinity at zero tension to a minimum at the tension of several millinewtons per meter. Transmembrane voltage, on the contrary, caused the waiting time to decrease monotonously. Analysis of pore formation trajectories for several lipid species with different spontaneous curvatures and elastic moduli under various external conditions provided instrumental insights into the mechanisms underlying some experimentally observed phenomena.
Lipid membranes serve as effective barriers allowing cells to maintain internal composition diffe... more Lipid membranes serve as effective barriers allowing cells to maintain internal composition differing from that of extracellular medium. Membrane permeation, both natural and artificial, can take place via appearance of transversal pores. The rearrangements of lipids leading to pore formation in the intact membrane are not yet understood in details. We applied continuum elasticity theory to obtain continuous trajectory of pore formation and closure, and analyzed molecular dynamics trajectories of pre-formed pore reseal. We hypothesized that a transversal pore is preceded by a hydrophobic defect: intermediate structure spanning through the membrane, the side walls of which are partially aligned by lipid tails. This prediction was confirmed by our molecular dynamics simulations. Conversion of the hydrophobic defect into the hydrophilic pore required surmounting some energy barrier. A metastable state was found for the hydrophilic pore at the radius of a few nanometers. The dependence of the energy on radius was approximately quadratic for hydrophobic defect and small hydrophilic pore, while for large radii it depended on the radius linearly. The pore energy related to its perimeter, line tension, thus depends of the pore radius. Calculated values of the line tension for large pores were in quantitative agreement with available experimental data.
The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kin... more The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein-lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated act...
Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic p... more Heat-activated transient receptor potential channel TRPV1 is one of the most studied eukaryotic proteins involved in temperature sensation. Upon heating, it exhibits rapid reversible pore gating, which depolarizes neurons and generates action potentials. Underlying molecular details of such effects in the pore region of TRPV1 is of a crucial importance to control temperature responses of the organism. Despite the spatial structure of the channel in both open (O) and closed (C) states is known, microscopic nature of channel gating and mechanism of thermal sensitivity are still poorly understood. In this work, we used unrestrained atomistic molecular dynamics simulations of TRPV1 (without N-and C-terminal cytoplasmic domains) embedded into explicit lipid bilayer in its O-and C-states. We found that the pore domain with its neighboring loops undergoes large temperature-dependent conformational transitions in an asymmetric way, when fragments of only one monomer move with large amplitude, freeing the pore upon heating. Such an asymmetrical gating looks rather biologically relevant because it is faster and more reliable than traditionally proposed "iris-like" symmetric scheme of channel opening. Analysis of structural, dynamic, and hydrophobic organization of the pore domain revealed entropy growth upon TRPV1 gating, which is in line with current concepts of thermal sensitivity.
Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family tran... more Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.
Abstract: Membrane and membrane-active peptides and proteins play a crucial role in numerous cell... more Abstract: Membrane and membrane-active peptides and proteins play a crucial role in numerous cell processes, such as signaling, ion conductance, fusion, and others. Many of them act as highly specific and efficient drugs or drug targets, and, therefore, attract ...
Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large-scale structural rearrangement... more Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large-scale structural rearrangements during spliceosome assembly, activation and catalysis. This is exemplified by the highly conserved U6 snRNA which can exist in isolation in the U6 small nuclear ribonucleoprotein (snRNP); basepaired with U4 in the U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and spliceosomal B complex; or basepaired with the pre-mRNA intron and the U2 snRNA in the catalytic spliceosome. Through genetic evidence and structure probing (1, 2), it was previously proposed that the U4/U6 di-snRNA consisted of a three-helical junction core domain flanked by single stranded regions. Using single-molecule Förster resonance energy transfer (smFRET), we discovered a dynamic equilibrium between RNA conformations formed by the flanking single stranded regions. Our smFRET data is consistent with the presence of two mutually exclusive structures: (1) a U4/U6 di-snRNA containing both basepaired U4/U6 and the U6 telestem and (2) a U4/U6 di-snRNA with extended basepairing located near U4/U6 stem I. These data show that the U6 telestem persists in the U4/U6 di-snRNA and that its formation can be regulated by novel basepairing interactions with U4. This suggests conservation of mechanism between the yeast and human U4/ U6 di-snRNAs. Finally, we speculate that telestem formation can be dynamically regulated by spliceosomal proteins to aid in tri-snRNP disassembly and spliceosome activation_rationalizing previous observations concerning the mechanism of the U4/U6 RNA helicase Brr2.
Specific interactions between transmembrane α-helices, to a large extent, determine the biologica... more Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions betwee...
Journal of bioinformatics and computational biology, 2007
Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc... more Structure activity relationships were investigated for membrane-lytic peptides (MLP) Ltc1 and Ltc2a from the latarcin family. The peptides were studied via long-term molecular dynamics (MD) simulations in different membrane environments (detergent micelles, mixed lipid bilayers mimiking eukaryotic and bacterial membranes). The calculated structure of Ltc2a in sodium dodecyl sulfate micelle agrees well with the data obtained by (1)H-NMR spectroscopy. This validates the applied modeling approach. The binding mode of MLPs is governed by several factors: (i) the membrane surface curvature; (ii) the conformational plasticity and hydrophobic organization of the peptide, which depend on the arrangement of charged, non-polar and helix-breaking residues in the amino acid sequence. In contrast to Ltc1, insertion of Ltc2a into model membranes induces significant changes in dynamic behavior of lipids in the contact region. Such a prominent membrane destabilization correlates with high membrane-...
Biochemical and biophysical research communications, Jan 7, 2014
Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammator... more Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.
Advances in protein chemistry and structural biology, 2011
This review surveys recent achievements of molecular computer modeling in understanding spatial s... more This review surveys recent achievements of molecular computer modeling in understanding spatial structure, dynamics, and mechanisms of functioning of transmembrane α-helical dimers in membranes. The factors driving self-association of hydrophobic helices in the membrane milieu are considered with examples of their applications to biologically relevant problems. The emphasis is made on the recent results, which help to understand important aspects of structure-function relations for these systems and their biological activity. Limitations and shortcomings of the methods, along with their perspectives in design of new membrane active agents, are discussed.
The hypothesis that local conformational differences of snake venom cardiotoxins (cytotoxins, CTs... more The hypothesis that local conformational differences of snake venom cardiotoxins (cytotoxins, CTs) may play a significant role in their interaction with membrane was tested by molecular modeling of the behavior of the CT A5 from the venom of Naja atra in water and at the water-membrane interface. Two models of the CT A5 spatial structure are known: the first was obtained by X-ray analysis and the second, by NMR studies in solution. A molecular dynamics (MD) analysis demonstrated that loop II of the toxin has a fixed omega-like shape in water, which does not depend on its initial structure. Interaction of the experimentally derived (X-ray and NMR) conformations and MD-simulated conformations of CT A5 with the lipid bilayer was studied by the Monte Carlo method using the previously developed model of the implicit membrane. The following was found: (1) Unlike the previously studied CT2 from the venom of cobra Naja oxiana, CT A5 has only loops I and II bound to the membrane, with the in...
unsupported bilayers. Recent experiments show that zwitterionic lipid bilayers supported on parti... more unsupported bilayers. Recent experiments show that zwitterionic lipid bilayers supported on partially charged, nanoporous silica wafers also do not interact with silica directly. However, in such cases the bilayer is separated from the silica substrate by a relative thicker (~1.5 nm) layer of solvent. We report here results from our MD simulation that reconcile these two seemingly disparate observations. We find that LJ nano-substrates whose porosity and surface hydroxyl density match those of nanoporous silica wafers do not increase the substrate-bilayer distance. Instead, the introduction of partial charge on the LJ substrate increases the substrate-bilayer distance to 1.5 nm. The negative charge on the substrate attracts hydrated ions, creating an electric double layer, which is a sufficient condition to increase the substrate-bilayer distance. While the properties of the bilayer supported on a charged substrate are also different from those of unsupported bilayers, the extent of the perturbation is not as large compared to that induced by uncharged hydroxylated substrates.
Ela®n, a speci®c inhibitor of elastase, is thought to play a regulatory role in in¯ammation. An N... more Ela®n, a speci®c inhibitor of elastase, is thought to play a regulatory role in in¯ammation. An NMR-derived solution structure of recombinant ela®n has been reported [Francart et al. (1997) J Mol Biol 268:666 ], although the conformation of its¯exible N-terminal part is not established. There is experimental evidence that the N terminus (residues 1±15) of ela®n interacts with the cell membrane. To explore the conformational preferences of residues in this region, we have performed Monte Carlo simulations of the peptide in water, in cyclohexane, and in a model membrane. Additionally, 3.7-ns molecular dynamics with explicit water was carried out. The main results were that the hydrophobic environment stabilizes an a helix in the region 6±11, the peptide is unordered in water, and it is attached to the membrane via the amphiphilic a-helix 6±11, which inserts with its N terminus forming an angle of about 60°to the membrane plane. We therefore assume that in nonpolar media the N-terminal part of ela®n forms a short a helix which might act as a membrane anchor.
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Papers by Pavel Volynsky