Papers by Andriy Anishkin
Symmetry improves ion channels stability into membrane
Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases... more Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5–50%) a...
Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases... more Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while
gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs.
The recA/RAD51 gene family encodes a diverse set of recombinase proteins that effect homologous r... more The recA/RAD51 gene family encodes a diverse set of recombinase proteins that effect homologous recombination, DNA-repair, and genome stability. The recA gene family is expressed in almost all species of Eubacteria, Archaea, and Eukaryotes, and even in some viruses. To date, efforts to resolve the deep evolutionary origins of this ancient protein family have been hindered, in part, by the high sequence divergence between families (i.e. ~30% identity between paralogous groups). Through (i) large taxon sampling, (ii) the use of a phylogenetic algorithm designed for measuring highly divergent paralogs, and (iii) novel Evolutionary Spatial Dynamics simulation and analytical tools, we obtained a robust, parsimonious and more refined phylogenetic history of the recA/RAD51 superfamily. Taken together, our model for the evolution of recA/RAD51 family provides a better understanding of ancient origin of recA proteins and multiple events leading to the diversification of recA homologs in eukaryotes, including the discovery of additional RAD51 sub-families.
Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate
The Journal of general physiology, 2015
Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K(+) channels and voltage-gated Ca(... more Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K(+) channels and voltage-gated Ca(2+) channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K(+) channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K(+) channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site...
Mathematical Modeling of Energy Coupling in Mitochondria in Frames of Proton-Chemical Hypothesis
Biofizika
The dependence of ATP-synthesis and mitochondria respiration rates on membrane potential was anal... more The dependence of ATP-synthesis and mitochondria respiration rates on membrane potential was analyzed in frames of proton-chemical hypothesis of oxidative phosphorylation. The mathematical and electrical models of the unit of energy coupling are proposed. Computer analysis of the mathematical model allowed to obtain the theoretical dependence of the rates of ATP synthesis and of the respiration on the membrane potential. Both of all are in good qualitative agreement with experimental data, contradicting chemiosmotic hypothesis.
Ceramide channels: destabilization by Bcl-xL and role in apoptosis
Biochimica et biophysica acta, 2015
Ceramide is a bioactive sphingolipid involved in mitochondrial-mediated apoptosis. Our data sugge... more Ceramide is a bioactive sphingolipid involved in mitochondrial-mediated apoptosis. Our data suggest that ceramides directly regulate a key initiation step in apoptosis: mitochondrial outer membrane permeabilization (MOMP). MOMP allows release of intermembrane space proteins to the cytosol, inducing the execution of the cell. Ceramides form channels in planar phospholipid membranes and outer membranes of isolated mitochondria, channels large enough to facilitate passage of proteins released during MOMP. Bcl-xL inhibits MOMP in vivo and inhibits the formation of ceramide channels in vitro. However the significance of Bcl-xL's regulation of ceramide channel formation within cells was untested. We engineered Bcl-xL point mutations that specifically affect the interaction between ceramide and Bcl-xL to probe the mechanism of ceramide channel regulation and the role of ceramide channels in apoptosis. Using these mutants and fluorescently-labeled ceramide, we identified the hydrophobic...
PNAS-2015-Teng-1502366112
Symmetry improves ion channels stability into membrane
Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases... more Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5–50%) a...
Mechanosensitive TReK channels belong to the family of K2p channels, a family of widely distribut... more Mechanosensitive TReK channels belong to the family of K2p channels, a family of widely distributed, well modulated channels that uniquely have two similar or identical subunits, each with two TM1-p-TM2 motifs. Our goal is to build viable structural models of TReK channels, as representatives of K2p channels family. The structures available to be used as templates belong to the 2TM channels superfamily. These have low sequence similarity and different structural features: four symmetrically arranged subunits, each having one TM1-p-TM2 motif. Our model building strategy used two subunits of the template (Kcsa) to build one subunit of the target (TReK-1). Our models of the Closed channel were adjusted to differ substantially from those of the template, e.g., TM2 of the 2 nd repeat is near the axis of the pore whereas TM2 of the 1 st repeat is far from the axis. segments linking the two repeats and immediately following the last TM segment were modeled ab initio as α-helices based on helical periodicities of hydrophobic and hydrophilic residues, highly conserved and poorly conserved residues and statistically related positions from multiple sequence alignments. The models were further refined by two-fold symmetry-constrained MD simulations using a protocol we developed previously. We also built models of the Open state and suggest a possible tension-activated gating mechanism characterized by helical motion with two-fold symmetry. Our models are consistent with deletion/truncation mutagenesis and thermodynamic analysis of gating described in the accompanying paper.
Journal of Molecular Biology, 2015
Type II bacterial L-asparaginases (L-ASP) have played an important therapeutic role in cancer tre... more Type II bacterial L-asparaginases (L-ASP) have played an important therapeutic role in cancer treatment for over four decades, yet their exact reaction mechanism remains elusive. L-ASP from E. coli deamidates asparagine (Asn) and glutamine, with a ~10 4 higher specificity (k cat /K m ) for asparagine despite only one methylene difference in length. Through a sensitive kinetic approach, we quantify competition among the substrates and interpret its clinical role. To understand specificity, we use molecular simulations characterize enzyme interactions with substrates and a product (aspartate). We present evidence that the aspartate product in the crystal structure of L-ASP exists in an unusual -COOH protonation state. Consequently, the set of enzyme-product interactions found in the crystal structure, which guided prior mechanistic interpretations, differs from those observed in dynamic simulations of the enzyme with the substrates. Finally, we probe the initial nucleophilic attack with ab initio simulations. The unusual protonation state reappears, suggesting that crystal structures (wild-type and a T89V mutant) represent intermediate steps rather than initial binding. Also, a proton transfers spontaneously to Asn, advancing a new hypothesis that the substrate's -carboxyl serves as a proton acceptor and activates one of the catalytic threonines during L-ASP's nucleophilic attack on the amide carbon. That hypothesis explains for the first time why proximity of the substrate -COOgroup to the carboxamide is absolutely required for catalysis. The substrate's catalytic role is likely the determining factor in enzyme specificity as it constrains the allowed distance between the backbone carboxyl and the amide carbon of any L-ASP substrate.
Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin
PloS one, 2015
The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been ful... more The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas th...
[Mathematical modeling of energy coupling in mitochondria in frames of proton-chemical hypothesis]
Biofizika
The dependence of ATP-synthesis and mitochondria respiration rates on membrane potential was anal... more The dependence of ATP-synthesis and mitochondria respiration rates on membrane potential was analyzed in frames of proton-chemical hypothesis of oxidative phosphorylation. The mathematical and electrical models of the unit of energy coupling are proposed. Computer analysis of the mathematical model allowed to obtain the theoretical dependence of the rates of ATP synthesis and of the respiration on the membrane potential. Both of all are in good qualitative agreement with experimental data, contradicting chemiosmotic hypothesis.
Theoretical Dependence of Phosphate Potential on Magnitude of Protonmotive Force for Two-Electron Proton-Chemical Coupling Point Model
Mathematical Simulation of Energy Coupling in Mitochondria within the Framework of the Proton–Chemical Hypothesis
Microbial mechanosensation
Nature Communications, 2014
Animals need to sense and react to potentially dangerous environments. TRP ion channels participa... more Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca 2 þ influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca 2 þ influx. Within the selectivity filter, M 601 -F 609 , Y604G strongly reduces avoidance behaviour and eliminates Ca 2 þ transients. Y604F also abolishes Ca 2 þ transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y 604 may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.
Beta-barrel models of soluble amyloid beta oligomers and annular protofibrils
Proteins: Structure, Function, and Bioinformatics, 2010
Both soluble and membrane-bound prefibrillar assemblies of Abeta (Aβ) peptides have been associat... more Both soluble and membrane-bound prefibrillar assemblies of Abeta (Aβ) peptides have been associated with Alzheimer's disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aβ42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six-stranded β-barrel formed by the last third of Aβ42, which is composed of hydrophobic residues and glycines. The hydrophobic core β-barrels of the hexameric models are shielded from water by the N-terminus and central segments. These more hydrophilic segments were modeled to have either predominantly β or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results.
Proceedings of the National Academy of Sciences, 2013
How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and... more How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and several eukaryotic channels were found to come from the lipid membrane. Our survey of animal cells found that membrane force foci all have cholesterol-gathering proteins and are reinforced with cholesterol. This result is evident in overt force sensors at the tips of stereocilia for vertebrate hearing and the touch receptor of Caenorhabditis elegans and mammalian neurons. For less specialized cells, cadherins sustain the force between neighboring cells and integrins between cells and matrix. These tension bearers also pass through and bind to a cholesterol-enriched platform before anchoring to cytoskeleton through other proteins. Cholesterol, in alliance with sphingomyelin and specialized proteins, enforces a more ordered structure in the bilayer. Such a stiffened platform can suppress mechanical noise, redirect, rescale, and confine force. We speculate that such platforms may be dynamic. The applied force may allow disordered-phase lipids to enter the platform-staging channel opening in the thinner mobile neighborhood. The platform may also contain specialized protein/lipid subdomains enclosing mechanosensitive channels to open with localized tension. Such a dynamic stage can mechanically operate structurally disparate channels or enzymes without having to tie them directly to cadherin, integrin, or other protein tethers.
Microbial mechanosensation
Current Opinion in Neurobiology, 2005
Because bacterial mechanosensitive channels have been cloned, purified, crystallized and subjecte... more Because bacterial mechanosensitive channels have been cloned, purified, crystallized and subjected to a genetic, biochemical and physical scrutiny, they have become the current structural models of mechanosensation to atomic detail. The key observation, supported by recent mutagenesis studies, is that these channels receive stretch force directly through the lipid bilayer at the interface levels bearing highest tension. Indeed, simulations of mechanosensitive channels steered by strategically applied bilayer stretch forces show channel opening. Our understanding of the gating energetics and trajectory are continually being refined by the combination of approaches applied. In addition, new microbial mechanosensitive channels from the TRP family have been characterized in yeasts. Unified by fundamental biophysical principles of gating, mechanosensitive channels provide broad insight into protein-membrane interactions and the role of hydrophobic hydration in gating.
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Papers by Andriy Anishkin
gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs.