The conformational states of Mg·ATP in water

The conformational equilibria of Mg·ATP in solution is studied using molecular dynamics (MD) augmented with umbrella sampling methods. Free energy comparisons show that the Mg²⁺ ion is equally likely to coordinate the oxygens of the two end phosphates, or of all three phosphates. The MD trajectories reveal two major degrees of freedom of the Mg·ATP molecule in solution, and we compute the free energy as a function of these variables, and determine its elastic properties. Comparing the free energy function with several crystallographic structures of ATP analogs, we find that the crystal structures correspond to states where ATP would be elastically strained. The average water density around Mg·ATP is investigated to show the average number of hydrogen bonds and the hydrophobicity.     

A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Ca_Vβ subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca_V2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca_V2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca_Vβ (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca_V2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca_V2.3 channels. Furthermore, the normalized protein density of Ca_V2.3 was nearly abolished with the quadruple Ca_Vβ3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca_Vβ3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca_V2.3. This interaction appears to play an essential role in conferring Ca_Vβ-induced modulation of the protein density of Ca_Vα1 subunits in Ca_V2 channels.     

The Role οf Ion Channels in the Development and Progression of Prostate Cancer

Molecular Diagnosis & Therapy - Ion channels have major regulatory functions in living cells. Apart from their role in ion transport, they are responsible for cellular electrogenesis and...     

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the β6-β7 loop that is essential for both maturation and function. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the β6-β7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function.     

The Properties of Ion Channels in Lipid Membranes Modified by the Aromatic Antibiotic Levorin А2

It has been shown that the main components of levorin A, that is, A₀, A₁, A₂, or A₃, that contain an aromatic group increase the permeability of membranes in the series A₃ > A₂ > A₁ > A₀ when they are on the same side of the membrane. All levorin components have cationic selectivity. The most studied levorin, А₂, promotes the almost ideal permeability of membranes to potassium ions. The membrane potential for a ten-fold change in the KCl concentration gradient is 56 ± 2 mV. It has been shown that the injection of the same concentration of levorin А₂ into one side of the membrane and then, after achieving the typical membrane permeability, into the other side of the membrane generates a two-fold increase in the total membrane permeability. This means that independent levorin-induced conductive semi-pores are formed on each side of the membrane. It has been found that the injection of levorin А₂ only into one side of the membrane enhances the membrane permeability to monosaccharides and other neutral molecules. The presence of levorin А₂ in cholesterol-, ergosterol-, and stigmasterol-containing phospholipid membranes has been shown to lead to the single-channel conductivity of typical ion channels of 0.2–0.5 pS. The properties of these channels have been studied. The levorin channels exist in two states, open and closed. Most of the time, the channel remains in the open state in the KBr solution. In solutions of different salts of the same concentration, the conductivity value of the levorin channels is approximately the same (0.4–0.5 pS). An increase in the dimethyl sulfoxide concentration in aqueous solutions facilitates the transition of polyene antibiotic molecules from dispersed to monomolecular form. The molecules of polyene antibiotics in the associated form exhibit high membrane activity.

     

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

1. Download : Download high-res image (314KB)
2. Download : Download full-size image

Highlights? Production of soluble NIK kinase domain ? Structures of apo murine and human NIK possess active conformation ? Structure of mNIK bound to inhibitors reveals conformational flexibility ? Inhibitor potency varies against mNIK and hNIK due to substitution in the active site     

The Role of SV Ion Channels Under the Stress of Mycotoxins Induced in Wheat Cells—Protective Action of Selenium Ions

Journal of Plant Growth Regulation - Mycotoxin stress, one of the currently intensively studied, was stimulated in wheat hypocotyl cells through the application of zearalenone, produced by Fusarium...     

The Protein Kinase C�� Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G₂/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G₂/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr¹⁵), a critical event in the G₂/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G₂/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G₂/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G₂/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G₂/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G₂/M cell cycle checkpoint may be a critical component of its tumor suppressor function.     

Effect of Activators and Blockers of Ligand-Regulated Ion Channels on the Activity of the Cl−-Stimulated Mg2+-ATPase of the Plasma Membrane Fraction from Bream (Abramis brama L.) Brain

Effects of GABA, glycine, acetylcholine, and glutamate (agonists of the GABA_a/benzodiazepine, glycine, choline, and glutamate receptors, respectively) at concentrations in the range 10^–8-10^–4 M on the activity of basal Mg²⁺-ATPase of the plasma membrane fraction from bream brain and on its activation by Cl^– were investigated. GABA and glycine activated basal Mg²⁺-ATPase activity and suppressed its activation by Cl^–. Acetylcholine and glutamate activated basal Mg²⁺-ATPase to a lesser extent and did not suppress the activation of the enzyme by Cl^–.The activation of basal Mg²⁺-ATPase by neuromediators was decreased by blockers of the corresponding receptors (picrotoxin, strychnine, benztropine mesylate, and D-2-amino-5-phosphonovaleric acid). In addition, picrotoxin and strychnine eliminated the inhibiting effect of GABA and glycine, respectively, on the Cl^–-stimulated Mg²⁺-ATPase activity. Agonists of the GABA_a/benzodiazepine receptor–phenazepam (10^–8-10^–4 M) and pentobarbital (10^–6-10^–3 M)–activated the basal Mg²⁺-ATPase activity and decreased the Cl^–-stimulated Mg²⁺-ATPase activity. The dependence of both enzyme activities on ligand concentration is bell-shaped. Moreover, phenazepam and pentobarbital increased the basal Mg²⁺-ATPase activity in the presence of 10^–7 M GABA and did not influence it in the presence of 10^–4 M GABA and 10^–6 M glycine. The data suggest that in the fish brain membranes the Cl^–-stimulated Mg²⁺-ATPase interacts with GABA_a/benzodiazepine and glycine receptors but not with m-choline and glutamate receptors.     

A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring

Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal ∼50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKG-anchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG Iβ dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.     

Solution Structure and Backbone Dynamics of the Pleckstrin Homology Domain of the Human Protein Kinase B (PKB/Akt). Interaction with Inositol Phosphates

The programmed cell death occurs as part of normal mammalian development. The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 3'-OH kinase (PI3K)/Protein Kinase B (PKB, also named Akt) cascade. Several targets of the PI3K/PKB signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote cell survival. PKB possesses a N-terminal Pleckstrin Homology (PH) domain that binds specifically and with high affinity to PtIns(3,4,5)P(3) and PtIns(3,4)P(2), the PI3K second messengers. PKB is then recruited to the plasma membrane by virtue of its interaction with 3'-OH phosphatidylinositides and activated. Recent evidence indicates that PKB is active in various types of human cancer; constitutive PKB signaling activation is believed to promote proliferation and increased cell survival, thereby contributing to cancer progression. Thus, it has been shown that induction of PKB activity is augmented by the TCL1/MTCP1 oncoproteins through a physical association requiring the PKB PH domain. Here we present the three-dimensional solution structure of the PH domain of the human protein PKB (isoform beta). PKBbeta-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a beta-sandwich of seven strands capped on one top by an alpha-helix. The opposite face presents three variable loops that appear poorly defined in the NMR structure. Measurements of (15)N spin relaxation times and heteronuclear (15)N[(1)H]NOEs showed that this poor definition is due to intrinsic flexibility, involving complex motions on different time scales. Chemical shift mapping studies correctly defined the binding site of Ins(1,3,4,5)P(4) (the head group of PtIns(3,4,5)P(3)), as was previously proposed from a crystallographic study. More interestingly, these studies allowed us to define a putative alternative low-affinity binding site for Ins(1,4,5)P(3). The binding of this sugar to PKBbeta-PH might also involve non-specific association that could explain the stabilization of the protein in solution in the presence of Ins(1,4,5)P(3).     

Brk/Protein Tyrosine Kinase 6 Phosphorylates p27KIP1, Regulating the Activity of Cyclin D–Cyclin-Dependent Kinase 4

Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27^Kip1 is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an “on/off switch.” We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.     

Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X7 Receptor: A Novel Conformation?

Scanning experiments have shown that the putative TM2 domain of the P2X₇ receptor (P2X₇R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X₂. These data led us to investigate a new architecture within the P2X receptor family. P2X₇R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X₇R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X₇R (hP2X₇R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X₇R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X₇R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X₇R field and/or raise new questions about this issue.     

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 ? resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.     

The Role of Dosage of the Region 7d1–7d5-6 of the X Chromosome in the Production of Homeotic Transformations in DROSOPHILA MELANOGASTER

A high frequency of homeotic transformations appears in Df(3)red/+ progeny of Df(1)sn^C128 /+ females. Generally, the metathoracic appendages are partially transformed into mesothoracic ones. Df(1)sn^C128 includes a small region of the X chromosome: 7D1 to 7D5-6. Hypodosage of this region is mainly effective at the level of the maternal genotype, and the effect is probably due to hypodosage of the wild-type allele of the gene fs(1)h. Df(3)red has an effect that is mainly, if not exclusively, zygotic, probably due to hypodosage of the wild-type allele of Rg-bx. The frequencies of transformed flies resulting from the interaction between Df(1)sn^C128 and Df(3)red are not very sensitive to external conditions and genetic background. Studies of the interactions between Df(1)sn^C128 and other mutations or deficiencies of chromosome 3 [Rg-pbx, bx, pbx, Ubx¹, Ubx¹³⁰, Ubx⁸⁰, Df(3)P9] reveal an analogy between the hypodosage effect of region 7D1–7D5-6 and the effects of ether treatment of blastoderm stage eggs. The role of the gene fs(1)h in the process of segment determination is discussed in the light of these results.