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The adenosine A.sub.1 receptor (A.sub.1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain.sup.1,2. However, development of analgesic orthosteric A.sub.1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects.sup.3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A.sub.1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A.sub.1R co-bound to adenosine, MIPS521 and a G.sub.i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A.sub.1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein., Author(s): Christopher J. Draper-Joyce [sup.1] [sup.10] , Rebecca Bhola [sup.2] , Jinan Wang [sup.3] , Apurba Bhattarai [sup.3] , Anh T. N. Nguyen [sup.1] , India Cowie-Kent [sup.2] , Kelly [...]

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments (1-7) and that lipids can bind to specific sites, for example, in potassium channels (8). Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation (9). AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 A resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding., Three membrane protein complexes were selected to give a range of topologies, oligomeric states and anticipated selectivity towards lipids: (1) the pentamericMscL from M. tuberculosis with two transmembrane helices per [...]

Allostery is the process by which biological macromolecules (mostly proteins) transmit the effect of binding at one site to another, often distal, functional site, allowing for regulation of activity. Recent experimental observations demonstrating that allostery can be facilitated by dynamic and intrinsically disordered proteins have resulted in a new paradigm for understanding allosteric mechanisms, which focuses on the conformational ensemble and the statistical nature of the interactions responsible for the transmission of information. Analysis of allosteric ensembles reveals a rich spectrum of regulatory strategies, as well as a framework to unify the description of allosteric mechanisms from different systems., Allostery, first articulated more than 50 years ago (1-3), has remained a central focus in biology, owing to the fact that a quantitative description of allostery is fundamental to an [...]

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1--S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and [Ca.sup.2+]-activated [K.sup.+] channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its 'decentralized' distribution of voltage-sensing residues, we probed the [BK.sub.Ca] VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization. BK | cooperativity | fluorometry | Slo1 | MaxiK doi/10.1073/pnas.0911959107

Allosteric signaling in proteins requires long-range communication mediated by highly conserved residues, often triggered by ligand binding. In this article, we map the allosteric network in the catalytic subunit of protein kinase A using NMR spectroscopy. We show that positive allosteric cooperativity is generated by nucleotide and substrate binding during the transitions through the major conformational states: apo, intermediate, and closed. The allosteric network is disrupted by a single site mutation (Y204A), which also decouples the cooperativity of ligand binding. Because protein kinase A is the prototype for the entire kinome, these findings may serve as a paradigm for describing long-range coupling in other protein kinases. allostery | NMR | signaling | enzymes | chemical shift mapping

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptor's binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with I[C.sub.50] [approximately equal to] 50 [micro]M and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3',5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3',5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target. antagonist | high-throughput screening | regulatory surface | antiandrogens | nuclear receptors

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking, in this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae. Vibrio vulnificus | VgrG | covalent actin cross-linking

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

The rotation of the [Gamma]-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase ([F.sub.0][F.sub.1]). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the y-subunit of the [F.sub.1] sector rotated on addition of ATP. This progress enabled us to analyze the [Gamma]M23K (the [Gamma]-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the [F.sub.1] sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the [Gamma]M23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

Oligo-directed mutagenesis, DNase I footprinting and in vivo transcription assays have been used in studying the regulatory region of the phoD promoter in Bacillus subtilis. Results indicate that a dimer of Pho-P can stably bind two consensus repeats while PhoP binding is highly cooperative within the core promoter region. It has also been found out that specific bases made up of TT(A/T/C)ACA consensus are necessary for transcriptional activation. The spacing between two consensus repeats within a putative dimer binding site in the core region is important for PhoP binding and promoter activation.

The binding of the anticancer agent, fagaronine (FGR), and ethoxidine (ETX), an inhibitor of DNA topoisomerase I and II, with DNA has been studied through Raman, surface-enhanced Raman scattering (SERS) and flow linear dichroism (FLD) spectroscopies. FGR was extracted from the roots of Fagara xanthoxyloides Lam while ETX was artificially synthesized. FLD data revealed that both compounds are strong major groove intercalators with stoichiometries 1 FGR/2.0 DNA bp and 1 ETX/4.0 DNA bp. The SERS spectrum of FGR was recorded at concentrations down to 10 raised to the -8th power and 10 raised to the -6th power for the spectrum of ETX.

The photo repair mechanism of DNA photolyase has been studied through molecular dynamics (MD) simulations. The investigation focuses on the cis,syn form, which is actively involved in the repair process. The docking and binding of bare and dressed model pyrimidine dimers U<>T and dUdT in the enzyme pocket have also been examined. The AMBER force field feature of the software Discover version 95.0 was used in doing the MD simulations. Results indicated that the dimers are inflexible and they have similar shapes. Van der Waals and Coulomb interactions were observed when the dimers dock into the enzyme pocket.

The ATP-sensitive potassium ([K.sub.ATP]) channels in pancreatic [Beta] cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of [K.sub.ATP] channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of [K.sub.ATP]-channel regulation remains unclear. The [K.sub.ATP] channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of [Gamma],-thio-ATP, stabilize the binding of prebound 8-azido-[[Alpha]-32]P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[[Alpha]-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.

Research was conducted to examine intramolecular hydrogen bonding in chloroform in small polyamides. Variable temperature infrared and nuclear magnetic resonance spectroscopies were used in the study of specific hydrogen bonds in selected triamides in organic solvents. Results indicate that each of the nine triamides studied exhibited its own unique intramolecular hydrogen-bonding pattern and that not all of the model peptides prefer the intramolecular hydrogen-bonded conformation.

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids 'sense' the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins ([Lambda] repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.

Research was conducted to evaluate the radical substituent effects of fluorine and trifluoromethyl groups from the computed C-H homolytic bond dissociation enthalpies of 30 fluorinated compounds. Results showed a strengthening of the sp(super 3) C-H bond with the introduction of three alpha-fluorine substituents. However, the entry of one or two substituents resulted in the weakening of C-H bond strength due to resonance delocalization.

Research was conducted to study the principle of maximum chiral discrimination. Molecular dynamics techniques were used to stimulate five guest molecules, namely, isomenthone, pulegone, 1-fluoro-1-phenylethane, 1-phenylethanol and 2-methylbutanoic acid, binding to permethyl-beta-cyclodextrin, a chiral host molecule. The study concludes that the host molecule exhibits its enhanced chiral discriminating capacity due to a spatial coincidence. The principle of maximum chiral recognition is demonstrated with the guest's preferred binding site.

The exo isomer of aflatoxin [B.sub.1] (AF[B.sub.1]) 8,9-epoxide appears to be the only product of AF[B.sub.1] involved in reaction with DNA and reacts with the [N.sup.7] atom of guanine via an [S.sub.N]2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in [H.sub.2]O (0.6 [s.sup.-1] at 25 [degrees] C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA ([K.sub.d] = 0.43 mg [ml.sup.-1], or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 [s.sup.-1]. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 [s.sup.-1] and half-maximal velocity at a DNA concentration of 1.8 mg [ml.sup.-1] (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 [s.sup.-1] at pH 6.2 with 0.17 mg DNA [ml.sup.-1]). The same rate enhancement effect was observed with poly[dA-dT][center dot]poly[dA-dT], in which the epoxide can intercalate but not form significant levels of [N.sup.7] purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells. LR1 also binds Ig heavy chain switch region sequences and may function in class switch recombination. LR1 contains two polypeptides, of 106 kDa and 45 kDa, and here we report that the 106-kDa component of LR1 is nucleolin. This identification, initially made by microsequence analysis, was verified by showing that (i) LR1-DNA binding activity increased in B cells transfected with a nucleolin cDNA expression construct; (ii) LR1-DNA binding activity was recognized by antibodies raised against recombinant human nucleolin; and (iii) in B cells transfected with epitope-tagged nucleolin expression constructs, the LR1-DNA complex was recognized by the anti-tag antibody. Nucleolin is an abundant nucleolar protein which is believed to play a role in rDNA transcription or organization, or rRNA processing. Homology between nucleolin and histone H1 suggests that nucleolin may alter DNA organization in response to cell cycle controls, and the nucleolin component of LR1 may therefore function to organize switch regions before, during, or after switch recombination. The demonstration that nucleolin is a component of a B cell-specific complex that binds switch region sequences suggests that the G-rich switch regions may have evolved from rDNA.

The heterologous transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression through cooperative binding to precisely juxtaposed DNA recognition elements. The molecular origins of cooperativity in the binding of NFAT and AP-1 to DNA are poorly understood. Herein we have used yeast one-hybrid screening and alanine-scanning mutagenesis to identify residues in AP-1 that affect cooperative interactions with NFAT on DNA. Mutation of a single conserved Arg residue to Ala in the c Jun spacer region (R285A) led to a virtually complete abolition of cooperative interactions with NFAT. The DNA-binding activity of AP-1 alone was unaffected by the cJun R285A mutation, thus indicating that this residue influences cooperative binding only. Ala-scanning mutations elsewhere in AP-1, including the cFos subunit, revealed no other strongly interacting single positions. We thus conclude that NFAT contacts AP-1 in the spacer region of the cJun subunit, making an especially important contact to R285, and that these interactions drive formation of the cooperative NFAT/AP-1/DNA complex. These results provide a general strategy for selectively ablating cooperativity between transcription factors without affecting their ability to act alone and yield insights into the structural basis for coordinate regulation of gene expression.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose.binding domain (CBD) from the [Beta]-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type [K.sub.a] of 6.3 ([+ or -] 1.4) x [10.sup.7] [M.sup.-1] and the low-affinity site by a [K.sub.a] of 1.1 ([+ or -] 0.6) x [10.sup.6] [M.sup.-1]. CB[D.sub.Cex] binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and [[Beta].sub.2]-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of [Zn.sup.2+]. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

The NusG-like protein from the hyperthermophilic bacterium Thermotoga maritima binds to DNA and RNA in a non-sequence-specific, generalized and highly cooperative manner. High levels of the protein affect in vitro transcription. The rate of DNA binding by the protein is the highest in the presence of approximately 50 mM salt. The DNA-protein complex does not get stained in ethidium bromide and resists cleavage by TaqI endonuclease. The complex contains approximately 30-40 NusG monomers/kb of duplex DNA. The RNA polymerase of T. maritima transcribes the NusG-like in vitro.

The fatty acylation and plasma membrane targeting characteristics of p59(super fyn) are responsible for the ability of the src homology (SH) 4 domain to bind to TCR proteins. Four amino acid residues are necessary for the proper binding of p59(super fyn) to TCR. The amino acids are present in p59(super fyn) and other src family of tyrosine kinases that bind to the immune-receptor tyrosine-based activation motif. The amino acids are also necessary for proper fatty acylation and plasma membrane targeting.

The nuclear architecture is disrupted by long-distance chromosome interactions that cause stochastic transcriptional silencing of genes that are present near heterochromatin (position-effect variegation). In the Drosophila embryonic nuclei, a heterochromatic insertion at the brown locus is spatially isolated from other heterochromatin. During larval development, this insertion physically associates with other heterochromatic regions on the same chromosome in a stochastic manner. The frequency of long distance interactions is highest in the interphase nuclei.

A method for identifying specific RNA-binding peptides using the arginine-rich RNA-binding motif as a framework is described. The arginine-rich motif may be a versatile framework for recognizing RNA structures. This was observed with the isolation of peptides from two combinatorial libraries that bind tightly and specifically to the Rev response element of HIV. Apparently, alpha-helical peptides resembling Rev were selected from one library while new peptides that probably do not form helices were selected from the other.

Cations bind to the Π face of an aromatic structure through a surprisingly strong, non-covalent force termed the cation-Π interaction. The magnitude and generality of the effect have been established by gas-phase measurements and by studies of model receptors in aqueous media. To first order, the interaction can be considered an electrostatic attraction between a positive charge and the quadrupole moment of the aromatic. A great deal of direct and circumstantial evidence indicates that cation-Π interactions are important in a variety of proteins that bind cationic ligands or substrates. in this context, the amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) can be viewed as polar, yet hydrophobic, residues., Noncovalent intermolecular forces play a major role in determining the structures of biological macromolecules and in mediating processes such as receptor-ligand interactions, enzyme-substrate binding, and antigen-antibody recognition. Although the hydrophobic [...]

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A study was conducted on receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) in vivo and in vitro in primed B and T cells. EtxB was compared with the receptor-binding mutant EtxB(G33D), where aspartate was substituted for glycine at residue 33. Results showed that the mutation Gly-33 - Asp triggered a significant reduction in the ability of EtxB to bind GM1 ganglioside.

The binding subunit of the interleukin-13 (IL-13) receptor which is also a part of the interleukin-4 (IL-4) receptor has been cloned and characterized. Data indicate that the IL-13 receptor alpha chain is the main binding subunit of the IL-13 receptor and could also be a component of IL-4 receptors. Murine NR4 was observed to encode IL-13Ralpha. while IL-13Ralpha expression was found to allow transduction of a proliferative signal by IL-13.

Insertion and deinsertion of the SecA subunit of the Escherichia coli preprotein translocase at SecYEG is controlled by ATP binding and the SecD and SecF proteins. The deinserted SecA, which is bound to SecYEG, inserts into the SecYEG membrane after preprotein binding to SecA. The energy for the insertion is supplied by the ATP binding to the nucleotide-binding domain 1 (NBD1). Hydrolysis of ATP causes the deinsertion of SecA. The SecA membrane cycling is also controlled by translocase interaction between SecD and SecF. The binding of preprotein to SecA induces protein secretion.

Binding of the transcription factor NF-kappa-B and the activating transfer factor ATF-2/c-Jun to the enhanceosome positive regulatory domains (PRDs) PRDII and PRDIV respectively changes the DNA curvature of the PRDs. The high mobility group protein HMG I(Y) alters the structure of these complexes. The conformational changes increase protein-protein interaction in the enhanceosome. The enhanceosome participates in the transcriptional activation of the interferon-beta gene in response to viral infection.

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), [p130.sup.Cas], and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of [p130.sup.Cas]. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse [p130.sup.Cas] amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that [p130.sup.Cas] and FAK are associated in mouse fibroblasts. The stable interaction between [p130.sup.Cas] and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Sre homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

The core proteins of large chondroitin sulfate proteoglycans contain a C-type lectin domain. The lectin domain of one of these proteoglycans, versican, was expressed as a recombinant 15-kDa protein and shown to bind to insolubilized fucose and GlCNAc. The lectin domain showed strong binding in a gel blotting assay to a glycoprotein doublet in rat brain extracts. The binding was calcium dependent and abolished by chemical deglycosylation treatment of the ligand glycoprotein. The versican-binding glycoprotein was identified as the cell adhesion protein tenascin-R, and versican and tenascin-R were both found to be localized in the granular layer of rat cerebellum. These results show that the versican lectin domain is a binding domain with a highly targeted specificity. It may allow versican to assemble complexes containing proteoglycan, an adhesion protein, and hyaluronan.

Nsp1p associates with the various nuclear pore complex (NPC) structures and is involved in multiple interactions at the NPC. The carboxy-terminal domain demonstrating heptad repeats of the nuclear pore protein, Nup82, binds to Nsp1p. In yeast cells where Nup82p is absent, cell growth is inhibited and a disorder is observed in the poly(A)+ RNA export.

The CD4 oligomerization on the cell surface is essential for obtaining a functional class II major histocompatibility complex (MHC) binding site, as explained by the unability to identify CD4 oligomers in the absence of class II MHC interaction by energy transfer methodology. Concentrating and crosslinking T-cell receptor (TCR) with its related signal transduction components, are possible by the network of CD4-class II MHC multimolecular complexes. For T-cell activation crosslinking of TCRs is critical.

The binding of the HIV envelope glycoprotein, gp120, to its host cell receptor, CD4, is inhibited in a solid phase assay by a glycosaminoglycan of human milk; this binding is the essential first step in HIV infectivity. The human milk glycosaminoglycans were identified in this study. Pooled, fractionated human milk contained dermatan sulfate, heparin, heparan sulfate, and chondroitin sulfate. The ability of this glycosaminoglycan fraction to inhibit binding was unaffected by digestion with lytic enzymes specific for heparin, heparan sulfate and dermatan sulfate, but was lost when the milk fraction was treated with lytic enzymes specific for chondroitin sulfate. Furthermore, a purified milk fraction with high specific inhibitory activity contained chondroitin sulfate but not other glycosaminoglycans. This indicates that the ability of human milk to inhibit gp120 binding to CD4 may be attributed to chondroitin sulfate or to a chondroitin sulfate-like moiety rather than to other components of human milk. We speculate that this human milk glycosaminoglycan could limit the rate of postnatal vertical transmission of HIV in breast-fed infants of HIV-infected mothers. INDEXING KEY WORDS: humans; milk; HIV; glycosaminoglycans; chondroitin sulfate

A study of the preferential binding of NikA protein to plasmid R64 through DNase I footprinting analyses and gel retardation studies confirms the attachment of NikA, the protein product of nikA gene of plasmid R64, to the right arm of 17-bp inverted repeat sequences. This binding occurs near the nick site inside the 44-bp oriT core sequence and causes the DNA to bend within the oriT sequence of the plasmid.

A multivalent radioligand having sulfoglucuronylneolacto (SGNL)-oligosaccharides covalently attached to a radioiodinated carrier protein, bovine serum albumin, was constructed. The radioligand was prepared to probe the presence of SGNL-oligosaccharide receptor in the peripheral nervous system (PNS). The properties of a complementary SGNL-specific binding site on PNS myelin is presented.

A study was conducted on the effects of tetracaine, procaine and QX314 on Ca2+ release by the sarcoplasmic reticulum (SR). Purified skeletal muscle Ca2+ release channel complex was incorporated into planar lipid bilayers and examined. Results indicate adding tetracaine andprocaine to the bilayer's cytoplasmic side or SR lumenal side caused a reduction in the activity of Ca2+- and ATP-gated channels. Both drugs inhibitedchannel activity through binding with at least two cooperativelty interacting high-affinity regulatory sites of the SR Ca2+ release channel.

When glycine (418) of Escherichia coli glutathione reductase, which is in a closely packed region of the dimer interface, is replaced with a bulky trytophan resudue, the enzyme becomes highly cooperative (Hill coefficient 1.76) for glutathione binding. The cooperativity is lost when the mutant subunit is hybridized with a wild-type subunit to createa heterodimer. The mutation appears to disrupt atomic packing at the dimer interface, which induces a change of kinetic mechanism. A single mutation in a region of the protein remote from the active site can thus act as a molecular switch to confer cooperativity on an enzyme., Many enzymes are oligomeric; of these, some exhibit standard Michaelis-Menten kinetics and others display homotropic or heterotropic cooperativity in ligand binding and a characteristically sigmoidal dependence of reaction velocity on [...]

The affinities of nitrogen regulator I (NR I) and of NRI-phosphate for single binding sites were compared with the affinities for sites 1 and 2 located upstream from gInAp2. The measurements were then correlated with the ability of NRI-phosphate to activate transcription of glnAp2. Results show that NRI is not enough for sufficient activation and that activation occurs only at higher NRI concentration. The phosphorylation of NRI is perceived to induce a conformational change in the N-terminal domains of theNRI-phosphate dimers which interact strongly with each other.

A binding event caused a shift in the energy landscape of a folding funnel, an action that reflect shifts in the population of the substrates. According to E Freire, residues, upon binding, exhibit significant changes that are far away from the binding sites. Freire also noted that binding sites have regions of high and low structural stability. These observations would help in predicting the local flexible regions involved in molecular binding, as long as the molecular structures are available.

It is not clear how transcription factors bound at distal enhancer and proximal promoter sequences cooperate to stimulate transcription in vivo. To distinguish between different models for the action of enhancer elements, we have directly measured DNA binding of the Drosophila activator zeste by in vivo UV crosslinking. Experiments in Drosophila embryos show that binding of zeste protein to either the proximal promoter of the Ultrabithorax (Ubx) gene or to a Ubx enhancer element does not require the presence of the other element. However, significant transcription is observed only when both elements are present and bound by zeste. The results indicate that stimulation by an enhancer can occur by a mechanism other than increasing the occupancy of an activator to binding sites near the start site of transcription.

A variable temperature 1H NMR study with a naphthyl group attached at the 3-hydroxy position of beta-cyclodextrin and adamantanecarboxylic acid (AdCA) forms the basis of a binding scheme similar to one recently proposed from fluorescence measurements with 2-(p-toluidino)-6-naphthalenesulfonate. 1H NMR spectra of the two compounds show the structures and interactions between the covalently modified cyclodextrin and the external hydrophobic guest. Spectral shifts indicate that complexation of AdCA takes place via the primary hydroxyl side.

The mechanism of cooperative channel gating is being investigated through the activation of the nucleotide gate component of these channels. One research team was able to study covalent activation of cyclic nucleotide-gated channels by using 8-p-azidophenyacylthio-cGMP in place of cyclic GMP. Other researchers have also characterized the way the nucleotide component regulates ion currents through the channels using the Hill equation with values lower than one for the Hill constant.

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation. KEY WORDS: potassium channel * cooperativity * fluorescence * voltage-sensing * gating

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated [Alpha], [Beta], [Gamma], and [Epsilon]. The DBL domain from the A4tres that binds ICAM-1 is DBL[Beta] type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBL[Beta] domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBL[Beta] domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

A basic limitation of drug development is the inability of traditional 'small-molecule' pharmaceuticals to target large protein interfaces, many of which are desirable drug targets. On page 182 of this [...]