DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the β subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii

The PsaF-deficient mutant 3bF of Chlamydomonas reinhardtii was used to modify PsaF by nuclear transformation and site-directed mutagenesis. Four lysine residues in the N-terminal domain of PsaF, which have been postulated to form the positively charged face of a putative amphipathic α-helical structure were altered to K12P, K16Q, K23Q, and K30Q. The interactions between plastocyanin (pc) or cytochrome c6 (cyt c6) and photosystem I (PSI) isolated from wild type and the different mutants were analyzed using crosslinking techniques and flash absorption spectroscopy. The K23Q change drastically affected crosslinking of pc to PSI and electron transfer from pc and cyt c6 to PSI. The corresponding second order rate constants for binding of pc and cyt c6 were reduced by a factor of 13 and 7, respectively. Smaller effects were observed for mutations K16Q and K30Q, whereas in K12P the binding was not changed relative to wild type. None of the mutations affected the half-life of the microsecond electron transfer performed within the intermolecular complex between the donors and PSI. The fact that these single amino acid changes within the N-terminal domain of PsaF have different effects on the electron transfer rate constants and dissociation constants for both electron donors suggests the existence of a rather precise recognition site for pc and cyt c6 that leads to the stabilization of the final electron transfer complex through electrostatic interactions.

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365

A colonization mutant of the efficient root-colonizing biocontrol strain Pseudomonas fluorescens WCS365 is described that is impaired in competitive root-tip colonization of gnotobiotically grown potato, radish, wheat, and tomato, indicating a broad host range mutation. The colonization of the mutant is also impaired when studied in potting soil, suggesting that the defective gene also plays a role under more natural conditions. A DNA fragment that is able to complement the mutation for colonization revealed a multicistronic transcription unit composed of at least six ORFs with similarity to lppL, lysA, dapF, orf235/233, xerC/sss, and the largely incomplete orf238. The transposon insertion in PCL1233 appeared to be present in the orf235/233 homologue, designated orf240. Introduction of a mutation in the xerC/sss homologue revealed that the xerC/sss gene homologue rather than orf240 is crucial for colonization. xerC in Escherichia coli and sss in Pseudomonas aeruginosa encode proteins that belong to the λ integrase family of site-specific recombinases, which play a role in phase variation caused by DNA rearrangements. The function of the xerC/sss homologue in colonization is discussed in terms of genetic rearrangements involved in the generation of different phenotypes, thereby allowing a bacterial population to occupy various habitats. Mutant PCL1233 is assumed to be locked in a phenotype that is not well suited to compete for colonization in the rhizosphere. Thus we show the importance of phase variation in microbe–plant interactions.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase

Methionine aminopeptidase (MetAP) exists in two forms (type I and type II), both of which remove the N-terminal methionine from proteins. It previously has been shown that the type II enzyme is the molecular target of fumagillin and ovalicin, two epoxide-containing natural products that inhibit angiogenesis and suppress tumor growth. By using mass spectrometry, N-terminal sequence analysis, and electronic absorption spectroscopy we show that fumagillin and ovalicin covalently modify a conserved histidine residue in the active site of the MetAP from Escherichia coli, a type I enzyme. Because all of the key active site residues are conserved, it is likely that a similar modification occurs in the type II enzymes. This modification, by occluding the active site, may prevent the action of MetAP on proteins or peptides involved in angiogenesis. In addition, the results suggest that these compounds may be effective pharmacological agents against pathogenic and resistant forms of E. coli and other microorganisms.

Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqα binds directly to the nonreceptor Bruton’s tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqα, not inactive GDP-bound Gqα, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqα also eliminate Btk stimulation by Gqα, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqα to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

Single prenyl-binding site on protein prenyl transferases

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.

Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei

In Trypanosoma brucei, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Three metal ions at the active site of the Tetrahymena group I ribozyme

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3′-oxygen of the oligonucleotide substrate and the 3′- and 2′-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity

Serotonin N-acetyltransferase is the enzyme responsible for the diurnal rhythm of melatonin production in the pineal gland of animals and humans. Inhibitors of this enzyme active in cell culture have not been reported previously. The compound N-bromoacetyltryptamine was shown to be a potent inhibitor of this enzyme in vitro and in a pineal cell culture assay (IC50 ≈ 500 nM). The mechanism of inhibition is suggested to involve a serotonin N-acetyltransferase-catalyzed alkylation reaction between N-bromoacetyltryptamine and reduced CoA, resulting in the production of a tight-binding bisubstrate analog inhibitor. This alkyltransferase activity is apparently catalyzed at a functionally distinct site compared with the acetyltransferase activity active site on serotonin N-acetyltransferase. Such active site plasticity is suggested to result from a subtle conformational alteration in the protein. This plasticity allows for an unusual form of mechanism-based inhibition with multiple turnovers, resulting in “molecular fratricide.” N-bromoacetyltryptamine should serve as a useful tool for dissecting the role of melatonin in circadian rhythm as well as a potential lead compound for therapeutic use in mood and sleep disorders.

The use of site-directed fluorophore labeling and donor–donor energy migration to investigate solution structure and dynamics in proteins

The use of molecular genetics for introducing fluorescent molecules enables the use of donor–donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor–donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the Cα-atoms of the positions V106C–H185C, H185C–M266C, and M266C–V106C were 60.9, 30.8, and 55.1 Å, respectively. These are in good agreement with the distances of 54 ± 4, 38 ± 3, and 55 ± 3 Å, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the Cα-atoms physically cannot coincide, there is a reasonable agreement between the methods.