Modeling the Calcium Signaling in Stomatal Guard Cells under the Action of Abscisic Acid

A theoretical model of calcium signaling is presented that simulates oscillations of cytoplasmic calcium concentration ([Ca²⁺]_cyt) in stomatal guard cells under the action of abscisic acid. The model is based on the kinetics of inositol 1,4,5-trisphosphate-sensitive calcium channels of endoplasmic reticulum and cyclic ADP-ribose-sensitive calcium channels of the tonoplast. The operation of two energy-dependent pumps—the Ca²⁺-ATPase of the endoplasmic reticulum and the Ca²⁺/H⁺ antiporter of the tonoplast—is also included in the model. It is shown that the removal of excessive Ca²⁺ from the cytoplasm by the tonoplast Ca²⁺/H⁺ antiporter is the main factor accounting for generation of [Ca²⁺]_cyt oscillations at a wide range of ABA concentrations (0.01–1 M). The long period of [Ca²⁺]_cyt oscillations in plant cells is explained by a slow release from inhibition of inositol 1,4,5-trisphosphate-gated calcium channels.     

A model of the dynamics of intracellular calcium based on refined kinetics of inhibiting the inositol-1,4,5-trisphoshate-sensitive calcium channel

Several new models of intracellular calcium dynamics based on refined inositol-1,4,5-triphosphate-sensitive calcium channel kinetics were studied. The refined kinetic schemes take into account that a cytosolic calcium cannot inhibit inositol-1,4,5-triphosphate receptors when they are bound to inositol-1,4,5-triphosphate. The mathematical analysis of intracellular calcium dynamics based on one of these schemes allowed us to show how different types of Ca response to extracellular stimuli, such as excitability, oscillations, sustained elevation of Ca and frequency encoding can arise with a reasonably good fit to experimental data.     

Bridging the gaps in 3D structure of the inositol 1,4,5-trisphosphate receptor-binding core

Calcium concentration is strictly regulated in all cells. The inositol 1,4,5-trisphosphate receptor (IP(3)R), which forms a homotetrameric Ca2+ release channel in the endoplasmic reticulum, is one of the key molecules responsible for this regulation. The opening of this channel requires binding of two intracellular messengers, which are inositol 1,4,5-trisphosphate (IP(3)) and Ca2+. To promote the Ca2+-channel gating and release from the endoplasmic reticulum, IP(3) binds to the amino-terminal region of IP(3)R. Recently, the crystal structure of IP(3)R-binding core in complex with its ligand was presented [I. Bosanac, J.R. Alattia, T.K. Mai, J. Chan, S. Talarico, F.K. Tong, K.I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba, M. Ikura, Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700; I. Bosanac, H. Yamazaki, T. Matsu-ura, T. Michikawa, K. Mikoshiba, M. Ikura, Crystal structure of the ligand-binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor, Mol. Cell 17 (2005) 193-203]. The space positions of residues 289-301 (segment A), 320-350 (segment B), 373-386 (segment C), and 529-545 (segment D) were not determined by the X-ray crystallography. To bridge these gaps, the computer modeling of physiologically meaningful low-energy 3D structures of the segments A-D of the inositol 1,4,5-trisphosphate receptor has been carried out by using a hierarchical conformational search algorithm combining two approaches: knowledge-based homology modeling and ab initio conformational search strategy. The structure analysis suggests a Ca2+-binding site of high affinity formed by residues 296-335, several low-energy regular secondary structure units within the segment B, and a number of hinge regions within the segments A-D, important for the receptor functioning.     

Monte Carlo simulations of tBid association with the mitochondrial outer membrane

Bid, a BH3-only pro-apoptopic member of the BCL-2 protein family, regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 α-helices (H1–H8, respectively) and is soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid by caspase 8 in apoptosis yields activated “tBid” (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. The release of cytochrome c from mitochondria to the cytosol constitutes a critical control point in apoptosis that is regulated by interaction of tBid protein with mitochondrial membrane. tBid displays structural homology to channel-forming bacterial toxins, such as colicins or transmembrane domain of diphtheria toxin. By analogy, it has been hypothesized that tBid would unfold and insert into the lipid bilayer of the mitochondria outer membrane (MOM) upon membrane association. However, it has been shown recently that unlike colicins and the transmembrane domain of diphtheria toxin, tBid binds to the lipid bilayer maintaining α-helical conformation of its helices without adopting a transmembrane orientation by them. Here, the mechanism of the association of tBid with the model membrane mimicking the mitochondrial membrane is studied by Monte Carlo simulations, taking into account the underlying energetics. A novel two-stage hierarchical simulation protocol combining coarse-grained discretization of conformational space with subsequent refinements was applied which was able to generate the protein conformation and its location in the membrane using modest computational resources. The simulations show that starting from NMR-established conformation in the solution, the protein associates with the membrane without adopting the transmembrane orientation. The configuration (conformation and location) of tBid providing the lowest free energy for the system protein/membrane/solvent has been obtained. The simulations reveal that tBid upon association with the membrane undergoes significant conformational changes primarily due to rotations within the loops between helices H4 and H5, H6 and H7, H7 and H8. It is established that in the membrane-bound state of tBid-monomer helices H3 and H5 have the locations exposed to the solution, helices H6 and H8 are partly buried and helices H4 and H7 are buried into the membrane at shallow depth. The average orientation of tBid bound to the membrane in the most stable configuration reported here is in satisfactory agreement with the evaluations obtained by indirect experimental means. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Activation of Bax by joint action of tBid and mitochondrial outer membrane: Monte Carlo simulations

The mitochondrial pathway of apoptosis proceeds when molecules, such as cytochrome c, sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane (MOM) permeabilization. Bax, a member of the Bcl-2 protein family, plays a pivotal role in mitochondrion-mediated apoptosis. In response to apoptotic stimuli, Bax integrates into the MOM, where it mediates the release of cytochrome c from the intermembrane space into the cytosol, leading to caspase activation and cell death. The pro-death action of Bax is regulated by interactions with both other prosurvival proteins, such as tBid, and the MOM, but the exact mechanisms remain largely unclear. Here, the mechanisms of integration of Bax into a model membrane mimicking the MOM were studied by Monte Carlo simulations preceded by a computer prediction of the docking of tBid with Bax. A novel model of Bax activation by tBid was predicted by the simulations. In this model, tBid binds to Bax at an interaction site formed by Bax helices α1, α2, α3 and α5 leading, due to interaction of the positively charged N-terminal fragment of tBid with anionic lipid headgroups, to Bax reorientation such that a hydrogen-bonded pair of residues, Asp98 and Ser184, is brought into close proximity with negatively charged lipid headgroups. The interaction with these headgroups destabilizes the hydrogen bond which results in the release of helix α9 from the Bax-binding groove, its insertion into the membrane, followed by insertion into the membrane of the α5–α6 helical hairpin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural Analysis of the HIV-1 gp120 V3 Loop: Application to the HIV-Haiti Isolates

Abstract

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent.

As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design.

The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.     

A theoretical analysis of pH dependence of sodium channel conductance

A new theory termed tunnel-acid-group-potential (TAGPT), which explains the effects of pHo and pHi on the ion conductance through different membrane channels, is presented. It is suggested that shifts in pHo and pHi change the values of negative charges generated by acid groups of side chains of some polar (Glu, Asp) amino acid residues lining the tunnel part of the channel. The resulting electrostatic field modification affects the heights of rate-limiting energy barriers (for ion transport) in the transition zones between the tunnel and the vestibules, which changes the channel conductance.     

The dynamics of the domains of the IP3-binding site of the inositol-1,4,5-triphosphate-sensitive calcium channel, induced by inositol-1,4,5-triphosphate and calcium

The dynamics of the inositol-1,4,5-triphosphate-sensitive calcium channel after binding of inositol-1,4,5-triphosphate and Ca2+ was analyzed by the Monte Carlo minimization technique. It was shown that the binding of Ca2+ with the unliganded receptor (channel) leads to a turning of the beta-sheet domain relative to the alpha-helical domain with the formation of the receptor conformation that is open for the entry of ions into the cytoplasmic channel vestibule, sterically closed for their passage through the vestibule in the part adjacent to the alpha-helical domains, and unfavourable for subsequent binding of inositol-1,4,5-triphosphate with the receptor. When both co-agonists bind to the receptor, the structure rearrangements induced eliminate both these steric obstacles for the passage of ions through the IP3-binding domain: one at the entrance of the channel cytoplasmic vestibule and the other that is placed deeper in the vestibule near the alpha-domains. The role of the dynamics of the receptor binding core in the IP3-sensitive channel gating is discussed.     

Structural analysis of the HIV-1 gp120 V3 loop: application to the HIV-Haiti isolates

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent. As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design. The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.     

Determination of structurally conservative amino acids of the HIV-1 protein gp120 V3 loop as promising targets for drug design by protein engineering approaches

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.