A regulator of G protein signaling interaction surface linked to effector specificity

Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the α subunits (Gα) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual Gα, and GTP hydrolysis rates of different Gαs can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-Gα binding interface and extends to include additional functionally important residues on the surface. One of these is within helix α3, two are in α5, and three are in the loop connecting α5 and α6. A cluster of surface residues on Gα previously identified by ET, and composed predominantly of residues from the switch III region and helix α3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the γ subunit of Gtα's effector, cGMP phosphodiesterase (PDEγ). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-Gα complex. Sequence variations in these residues correlate with PDEγ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the α-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Interaction of the poliovirus receptor with poliovirus

The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-Å resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus “canyon” and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is ΔU2SHNS,6SI2SHNS,6SI2SHNS,6SIHNAc,6SGHNS,3S,6S (±DDD4–7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

Gene-experience interaction alters the cholinergic septohippocampal pathway of mice

Spatial learning requires the septohippocampal pathway. The interaction of learning experience with gene products to modulate the function of a pathway may underlie use-dependent plasticity. The regulated release of nerve growth factor (NGF) from hippocampal cultures and hippocampus, as well as its actions on cholinergic septal neurons, suggest it as a candidate protein to interact with a learning experience. A method was used to evaluate NGF gene-experience interaction on the septohippocampal neural circuitry in mice. The method permits brain region-specific expression of a new gene by using a two-component approach: a virus vector directing expression of cre recombinase; and transgenic mice carrying genomic recombination substrates rendered transcriptionally inactive by a “floxed” stop cassette. Cre recombinase vector delivery into transgenic mouse hippocampus resulted in recombination in 30% of infected cells and the expression of a new gene in those cells. To examine the interaction of the NGF gene and experience, adult mice carrying a NGF transgene with a floxed stop cassette (NGFXAT) received a cre recombinase vector to produce localized unilateral hippocampal NGF gene expression, so-called “activated” mice. Activated and control nonactivated NGFXAT mice were subjected to different experiences: repeated spatial learning, repeated rote performance, or standard vivarium housing. Latency, the time to complete the learning task, declined in the repeated spatial learning groups. The measurement of interaction between NGF gene expression and experience on the septohippocampal circuitry was assessed by counting retrogradely labeled basal forebrain cholinergic neurons projecting to the hippocampal site of NGF gene activation. Comparison of all NGF activated groups revealed a graded effect of experience on the septohippocampal pathway, with the largest change occurring in activated mice provided with repeated learning experience. These data demonstrate that plasticity of the adult spatial learning circuitry can be robustly modulated by experience-dependent interactions with a specific hippocampal gene product.

Structural evidence for a programmed general base in the active site of a catalytic antibody

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation–π interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.

Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae

The Snf1 protein kinase family has been conserved in eukaryotes. In the yeast Saccharomyces cerevisiae, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation. The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal. Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1. Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system. This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site. The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a reg1Δ mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain. Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1. We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state.

Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is ≈35Å away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A–substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A’s ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein–protein interaction distant from the active site is critical for phosphorylation by cdks.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⋅substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

An important 2′-OH group for an RNA–protein interaction

We have investigated the role of 2′-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNACys and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2′-OH by 2′-deoxy or 2′-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2′-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2′-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2′-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2′-OH group instead provides a monitor of the steric environment during the RNA–synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2′-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations

rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA–protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem–loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem–loop IIa can participate in spliceosome assembly.

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.