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.

Ash1p is a site-specific DNA-binding protein that actively represses transcription

ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

Maintenance of Epstein–Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EBNA-1 amino acids 1–378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.

Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix–turn–helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors α- and γ-mediated gene expression via liver fatty acid binding protein: A signaling path to the nucleus

Peroxisome proliferator-activated receptor α (PPARα) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPARα colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPARα. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPARα and PPARγ but not with PPARβ and retinoid X receptor-α by protein–protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. Final and quantitative proof for L-FABP mediation was obtained in transactivation assays upon incubation of transiently and stably transfected HepG2 cells with saturated, monounsaturated, and polyunsaturated fatty acids as well as with hypolipidemic drugs. With all ligands applied, we observed strict correlation of PPARα and PPARγ transactivation with intracellular concentrations of L-FABP. This correlation constitutes a nucleus-directed signaling by fatty acids and hypolipidemic drugs where L-FABP acts as a cytosolic gateway for these PPARα and PPARγ agonists. Thus, L-FABP and the respective PPARs could serve as targets for nutrients and drugs to affect expression of PPAR-sensitive genes.

Competition for Microtubule-binding with Dual Expression of Tau Missense and Splice Isoforms

How tau mutations lead to neurodegeneration is unknown but may be related to altered microtubule binding properties of mutant tau protein. The tendency for the mutations to cluster around the microtubule-binding domain of tau or to alter the ratios of those splice isoforms that affect binding supports the view that the tau/microtubule interaction is critical and finely regulated. In cells transfected with both mutant and wild-type tau isoforms fused to either yellow fluorescent protein or cyan fluorescent protein we can observe tau fusion proteins that differ by a single amino acid or by the inclusion or exclusion of exon 10. With coexpression of mutant and wild-type tau, the mutant isoform appears diffuse throughout the cytoplasm; however, when mutant tau is expressed alone, it appears mostly bound to the microtubules. Dual imaging of the three- and four-repeat tau isoforms indicated that the expression of four-repeat tau displaced three-repeat tau from the microtubules. These results suggest that altered kinetic competition among the isoforms for microtubule binding could be a disease precipitant.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

To bind at an enzyme’s active site, a ligand must diffuse or be transported to the enzyme’s surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as “ionic tethering.” We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme’s surroundings even when the substrate is nonpolar.

Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion

Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single “fully competent” LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an α-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/ΔD1–2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/ΔD1–2 and with an ≈2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/ΔD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/ΔD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, “fully competent” LFA-1-binding surface.

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurring in vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding site per se are not essential for the TAP catalytic cycle.

A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses1

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.

Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.