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Selection for amino acid affinity by elution of RNAs from tryptophan-Sepharose using free L-tryptophan evokes one sequence predominantly (K(D) = 12 microM), a symmetrical internal loop of 3 nt per side. Though we have also isolated larger sequences with affinity for tryptophan, successively squeezed selection in randomized tracts of 70, 60, 40, 20 and 17 nt show that this internal loop is the simplest sequence that can meet the column affinity selection. From sequence variation in approximately 50 independent isolates, only 26 bits of information are required to describe this loop (equivalent to only 13 fully conserved nucleotides). Thus, it is among the simplest amino acid binding sites known, as well as selective among hydrophobic side chains. Among site sequences defined as essential to affinity by conservation, protection and modification-interference, there is a recurring CCA sequence (a tryptophan anticodon triplet) which apparently forms one side of the binding site. Such conserved juxtaposition of tryptophan with a cognate coding triplet supports a stereochemical origin for the genetic code.

The extracellular Ca2+-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca2+-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.

Aim: To design and synthesize a series of novel amino acid-binding 1,5-diarylpyrazole derivatives, which are intended to act as prodrugs with better aqueous solubility than celecoxib, and which will exert potent anti-inflammatory activities after being converted to their parent compounds in vivo . Methods: To introduce an amino acid, celecoxib analogs containing amino or methylamino group were synthesized first through multi-step chemical reactions. All the synthesized compounds were screened in an intact cell-based assay in vivo and in carrageenan-induced mouse paw edema in vivo . Some active compounds were selected for further evaluation in a carrageenan-induced rat paw edema model. The preliminary pharmacokinetics experiments were conducted using high performance liquid chromatography/mass spectrometry (HPLC/MS). Results: Celecoxib, 6 of the 1,5-diarylpyrazole class of celecoxib analogs, and their amino acid derivatives (hydrochloride salts) were synthesized. in vivo screening, the hydrochloride salts showed decreased inhibitory effects on cyclooxygenase (COX)-1 and COX-2 compared with their parent compounds, but some exhibited potent anti-inflammatory activity in vivo . Compound 4a was selected for further evaluation, and its anti-inflammatory effect was equivalent to that of celecoxib after oral administration in the carrageenan-induced rat paw edema model. At three doses (25 mg/kg, 50 mg/kg, and 100 mg/kg) the percentage inhibition on edema was 20.7%, 52.6%, and 62.6% (for compound 4a) and 27.8%, 38.4%, and 40.1% (for celecoxib), respectively. Preliminary pharmacokinetic evaluations support the hypothesis that compound 4a was actually converted to its parent compound, compound 4. Conclusion: The compound bound with amino acid acts like prodrug, which can exert anti-inflammatory effect similar to celecoxib after being converted to its parent compound. This finding will be of great benefit in carrying out structural modifications of prodrug-like selective COX-2 inhibitors.

The synthesis and binding properties of a new guanidiniocarbonyl pyrrole dication 2 are reported, which efficiently binds alanine carboxylate with log K ass = 3.9 in buffered water. Due to the increased charge density in this dication, the binding constant is five times larger than for the parent guanidiniocarbonyl pyrrole monocation 1 (log K = 3.2). However, the experimental conditions for determining the binding constant significantly influence both complex stability and stoichiometry. With increasing amount of substrate added during the titration, the overall complex stability decreases due to the increasing ionic strength of the solution. Furthermore, the formation of 1:2 complexes between 2 and 7 becomes increasingly important. Therefore, for the comparison of binding data it has to be assured that exactly the same experimental conditions are used for their determination.

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by l-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by l-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to l-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained l-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for l-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of l-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for l-glutamate is conserved as an l-amino acid binding site in its homolog, the CaR.

A relatively unexploited potential target for antimicrobial agents is the biosynthesis of essential amino acids. Homoserine dehydrogenase, which reduces aspartate semi-aldehyde to homoserine in a NAD(P)H-dependent reaction, is one such target that is required for the biosynthesis of Met, Thr, and Ile from Asp. We report a small molecule screen of yeast homoserine dehydrogenase that has identified a new class of phenolic inhibitors of this class of enzyme. X-ray crystal structural analysis of one of the inhibitors in complex with homoserine dehydrogenase reveals that these molecules bind in the amino acid binding region of the active site and that the phenolic hydroxyl group interacts specifically with the backbone amide of Gly175. These results provide the first nonamino acid inhibitors of this class of enzyme and have the potential to be exploited as leads in antifungal compound design.

In a continuing effort to unravel the structural basis for isoform-selective inhibition of nitric oxide synthase (NOS) by various inhibitors, we have determined the crystal structures of the nNOS and eNOS heme domain bound with two D-nitroarginine-containing dipeptide inhibitors, D-Lys-D-Arg(NO)2-NH(2) and D-Phe-D-Arg(NO)2-NH(2). These two dipeptide inhibitors exhibit similar binding modes in the two constitutive NOS isozymes, which is consistent with the similar binding affinities for the two isoforms as determined by K(i) measurements. The D-nitroarginine-containing dipeptide inhibitors are not distinguished by the amino acid difference between nNOS and eNOS (Asp 597 and Asn 368, respectively) which is key in controlling isoform selection for nNOS over eNOS observed for the L-nitroarginine-containing dipeptide inhibitors reported previously [Flinspach, M., et al. (2004) Nat. Struct. Mol. Biol. 11, 54-59]. The lack of a free alpha-amino group on the D-nitroarginine moiety makes the dipeptide inhibitor steer away from the amino acid binding pocket near the active site. This allows the inhibitor to extend into the solvent-accessible channel farther away from the active site, which enables the inhibitors to explore new isoform-specific enzyme-inhibitor interactions. This might be the structural basis for why these D-nitroarginine-containing inhibitors are selective for nNOS (or eNOS) over iNOS.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6–25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.

Aminoacyl-tRNA synthetases (aaRSs) strictly discriminate their cognate amino acids. Some aaRSs accomplish this via proofreading and editing mechanisms. Mursinna and coworkers recently reported that substituting a highly conserved threonine (T252) with an alanine within the editing domain of Escherichia coli leucyl-tRNA synthetase (LeuRS) caused LeuRS to cleave its cognate aminoacylated leucine from tRNA(Leu) (Mursinna et al., Biochemistry 2001;40:5376-5381). To achieve atomic level insight into the role of T252 in LeuRS and the editing reaction of aaRSs, a series of molecular modeling studies including homology modeling and automated docking simulations were carried out. A 3D structure of E. coli LeuRS was constructed via homology modeling using the X-ray structure of Thermus thermophilus LeuRS as a template because the E. coli LeuRS structure is not available from X-ray or NMR studies. However, both the X-ray T. thermophilus and homology-modeled E. coli structures were used in our studies. Amino acid binding sites in the proposed editing domain, which is also called the connective polypeptide 1 (CP1) domain, were investigated by automated docking studies. The root mean square deviation (RMSD) for backbone atoms between the X-ray and homology-modeled structures was 1.18 A overall and 0.60 A for the editing (CP1) domain. Automated docking studies of a leucine ligand into the editing domain were performed for both structures: homology structure of E. coli LeuRS and X-ray structure of T. thermophilus LeuRS for comparison. The results of the docking studies suggested that there are two possible amino acid binding sites in the CP1 domain for both proteins. The first site lies near a threonine-rich region that includes the highly conserved T252 residue, which is important for amino acid discrimination. The second site is located in a flexible loop region surrounded by residues E292, A293, M295, A296, and M298. The important T252 residue is at the bottom of the first binding pocket.

A detailed investigation of the nature of the binding of each of the 20 common alpha-amino acids and various selected dipeptides to a chiral, diaqua-ytterbium complex in aqueous solution has been carried out. Analysis of the dipolar 1H NMR paramagnetic shifts suggests that the alpha-amino acids form a common chelated structure within a nine-coordinate mono-capped square antiprismatic coordination environment, with the amine N axially disposed. Crystal structures of nine chelated YbL1-amino acid adducts (Gly, Ala, Ser, Thr, Met) confirm this. The ternary complexes with dipeptides (e.g. Gly-Ala, Gly-Ser, Gly-Met, Gly-Asp, Gly-Asn, Gly-His, Ser-Met, Asp-Phe, His-Gly) also favour the terminal amine as the axial donor with the proximate amide group binding to generate a five-ring chelate. Evidence for chelation through side-chain functionality was found only in the case of N-terminal Asp. The chiral environment about the ytterbium ion upon amino acid binding has also been probed using near-IR circular dichroism spectroscopy.

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA–amino acid binding structures.

Aminoacyl-tRNA synthetases guard the fidelity of cognate amino acid incorporation during protein biosynthesis; for example, phenylalanyl-tRNA synthetase (PheRS) activates and transfers only Phe to its tRNA. Since we are interested in using a computational protocol to identify nonnatural amino acids that are incorporated by wild-type PheRS, it is critical to understand the fidelity of PheRS in binding the 20 natural amino acids. To this end, HierDock, a computational protocol for predicting binding sites and relative binding affinities, was used for testing the natural amino acids in PheRS. Scanning the entire ligand-accessible protein surface for the best binding region, we find that HierDock correctly identifies the active site of Phe in PheRS and predicts Phe within 0.61 Å RMSD of the crystal structure. HierDock also successfully shows PheRS discriminates for Phe, as the noncognate amino acids bind less favorably in the binding site of Phe. However, we find that Met, Cys, and Tyr bind competitively but at positions distant from the Phe binding site. This result corroborates in vitro measurements of aminoacyl adenylate formation, which show Met competes with Phe at the amino acid binding stage. We predict that the binding site of Met would not activate PheRS, as the noncognate amino acid cannot establish suitable hydrogen bonds with the PheRS reaction center. These results validate the use of HierDock in predicting the binding sites of the cognate amino acids in PheRS. The HierDock procedure calculates the discrimination of aminoacyl-tRNA synthetases at the stage of binding the cognate amino acid and offers a molecular level understanding of the mistakes made in protein biosynthesis that are not readily uncovered through experiments. This technique is also useful for predicting the binding of a selected nonnatural amino acid analogue, thereby indicating whether the molecule would be incorporated into a wild-type aminoacyl-tRNA synthetase.

A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation. The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins. Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity. Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli. A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity. Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days. Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity. Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein. The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E. coli.

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) is a key player in Ca(2+)(o) homeostasis. The activity of CaR can be potentiated by various l-amino acids. In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by l-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC(50) for stimulation by Ca(2+)(o) and a modest increase in its maximal activity. In addition, mutating Ser-169 and Ser-171 together with Ser-170 yielded a more complete block of the phenylalanine modulation than did the single mutation. The presence of the triple mutation, S169A/S170A/S171A, also eliminated phenylalanine potentiation of the activities of heterodimeric receptors in which one of the monomeric receptors had intact triple serines (A877Stop). The putative amino acid binding site of the CaR is probably close to or structurally dependent on the Ca(2+)(o) binding sites of the receptor, because mutant CaRs with mutations in the putative amino acid binding site exhibited severely reduced responses to Ca(2+)(o).

We isolated RNAs by selection-amplification, selecting for affinity to Phe-Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The KD for the strongest sites is approximately 50 microM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 x 10(-11). Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA-amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.

γ-Glutamylcysteine synthetase (γ-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic γ-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial γ-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coliγ-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli γ-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli γ-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli γ-GCS binds two divalent metal ions;K d values for Mn2+ are 1.1 μm and 82 μm, respectively. Binding ofl-glutamate or l-BSO to the two Mn2+/γ-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the γ-GCS active site. The results are discussed in the context of the catalytic mechanism of γ-GCS and its relationship to the more fully characterized glutamine synthetase reaction.

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNA(Gln) for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNA(Gln) and tRNA(Glu) with glutamate. This ancient GluRS also separately differentiated to exclude tRNA(Gln) as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNA(Gln) and tRNA(Glu) recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.

We tested the substrate range of four wild-type Escherichia coli aminoacyl-tRNA synthetases (AARSs) with a library of non-standard amino acids (nsAAs). While these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also showed E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase to have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge to the goal of incorporating multiple different amino acids site-specifically into proteins.

Specific amino acid binding by aminoacyl-tRNA synthetases (aaRS) is necessary for correct translation of the genetic code. Engineering a modified specificity into aminoacyl-tRNA synthetases has been proposed as a means to incorporate artificial amino acid residues into proteins in vivo. In a previous paper, the binding to aspartyl-tRNA synthetase of the substrate Asp and the analogue Asn were compared by molecular dynamics free energy simulations. Molecular dynamics combined with Poisson-Boltzmann free energy calculations represent a less expensive approach, suitable for examining multiple active site mutations in an engineering effort. Here, Poisson-Boltzmann free energy calculations for aspartyl-tRNA synthetase are first validated by their ability to reproduce selected molecular dynamics binding free energy differences, then used to examine the possibility of Asn binding to native and mutant aspartyl-tRNA synthetase. A component analysis of the Poisson-Boltzmann free energies is employed to identify specific interactions that determine the binding affinities. The combined use of molecular dynamics free energy simulations to study one binding process thoroughly, followed by molecular dynamics and Poisson-Boltzmann free energy calculations to study a series of related ligands or mutations is proposed as a paradigm for protein or ligand design. The binding of Asn in an alternate, “head-to-tail” orientation observed in the homologous asparagine synthetase is analyzed, and found to be more stable than the “Asp-like” orientation studied earlier. The new orientation is probably unsuitable for catalysis. A conserved active site lysine (Lys198 in Escherichia coli) that recognizes the Asp side-chain is changed to a leucine residue, found at the corresponding position in asparaginyl-tRNA synthetase. It is interesting that the binding of Asp is calculated to increase slightly (rather than to decrease), while that of Asn is calculated, as expected, to increase strongly, to the same level as Asp binding. Insight into the origin of these changes is provided by the component analyses. The double mutation (K198L,D233E) has a similar effect, while the triple mutation (K198L,Q199E,D233E) reduces Asp binding strongly. No binding measurements are available, but the three mutants are known to have no ability to adenylate Asn, despite the “Asp-like” binding affinities calculated here. In molecular dynamics simulations of all three mutants, the Asn ligand backbone shifts by 1–2 A compared to the experimental Asp:AspRS complex, and significant side-chain rearrangements occur around the pocket. These could reduce the ATP binding constant and/or the adenylation reaction rate, explaining the lack of catalytic activity in these complexes. Finally, Asn binding to AspRS with neutral K198 or charged H449 is considered, and shown to be less favorable than with the charged K198 and neutral H449 used in the analysis.

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.

The amino acid binding properties of membrane fractions prepared from carp taste epithelia were compared to the results with those obtained in electrophysiological experiments. Electrophysiological cross-adaptation experiments using 12 amino acids showed that receptive mechanisms for these chemicals were able to be classified into six categories. The amount of binding of each amino acid was dose-dependent. Ligand bindings were also specific for membrane fractions of the taste epithelia, because the binding to those from carp head skin without taste buds was negligible. The binding of each amino acid indicates the existence of specific taste receptor binding sites in the facial taste system of the carp. However, there were some cases in which ligands competitively inhibited the binding of the parent amino acids, e. g., L-cysteine affected the binding of almost all the amino acid tested. Although this binding experiment itself could not totally explain the specificity of amino acid responses with electrophysiology, this specificity of response properties from this study could indicate that each ligand binds to specific receptor sites on the taste cell membrane.

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.

The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively. Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit. The conformations and binding of Phe are almost identical in both complexes. The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds. The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other. The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket. In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe. The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases. The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme. The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface. The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues. This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity.

Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity. When one of these residues, K89, which hydrogen-bonds to the γ-carboxyl group of the substrate l -glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine. In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum . Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates. The three-dimensional crystal structure of the GluDH from C. symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate. A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction. It has previously been proposed that this conformational change is triggered by substrate binding. However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate. Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate.

With this work we have completed the characterization of the syringomycin synthetase gene cluster. In particular, by sequencing additional 28.5 kilobase pairs we show that the nine modules involved in the binding of the nine amino acids of syringomycin are localized on SyrB and SyrE, with SyrE carrying eight modules. The recombinant SyrB and the first and second modules of SyrE (SyrE1 and SyrE2) have been expressed in Escherichia coliand purified. The biochemical data indicate that SyrB binds threonine, the putative precursor of the last amino acid of syringomycin, whereas SyrE1 and SyrE2 bind serine, the first and the second amino acids of syringomycin, respectively. On the basis of the sequence analysis and the biochemical data presented here, it appears that syringomycin synthetase is unique among peptide synthetases in that its genetic organization does not respect the “colinearity rule” according to which the order of the amino acid binding modules along the chromosome parallels the order of the amino acids on the peptide. This feature, together with the absence of a single transcription unit and the absence of epimerase-like domains make syringomycin synthetase more related to the eukaryotic peptide synthetases than to the bacterial counterparts.

Certain Bacillus licheniformis strains isolated from oil wells have been shown to produce a very effective biosurfactant, lichenysin A, which is structurally similar to another less active lipopeptide, surfactin. Surfactin, like many small peptides in prokaryotes and lower eukaryotes, is synthesized non-ribosomally by multi-enzyme peptide synthetase complex. Analysis of several peptide synthetases of bacterial and fungal origin has revealed a high degree of sequence conservation. Two 35-mer oligonucleotides derived from highly conserved motifs ('core I' and 'core II') of surfactin synthetase were used to identify the cloned putative operon of lichenysin A synthetase lchA from B. licheniformis BNP29, a strain not amenable to genetic manipulation in a BAC system (F-plasmid-based bacterial artificial chromosome) based on Escherichia coli and its single-copy plasmid F-factor. A 32.4 kb fragment containing lichenysin A biosynthesis locus was sequenced and analysed. The structural architecture of putative lichenysin A synthetase protein containing seven amino acid (aa) activation-thiolation, two epimerization and one thioesterase domains is discussed in terms of its similarity to surfactin and other peptide synthetases. The 100 aa peptide chain situated between the highly conserved signature sequences FDXX and NXYGPTE(IV)X within amino acid binding domains of peptide synthetases is proposed to be a minimal block dictating the substrate specificity of the enzymes. A new operon-type structure has been localized directly upstream from the lichenysin A synthetase genes which, on the basis of sequence determination, potentially encode a four-member ABC-type transport system involved in product secretion.

Formate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA- persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA-QB--QA QB-, in the direction of [QA-], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA- to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (approximately 70% of the wild type) level. (6) The Fo level was elevated (approximately 40-50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA-] in the dark. (7) A decreased (approximately 10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants.

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

Specific amino acid binding by aminoacyl-tRNA synthetases is necessary for correct translation of the genetic code. To obtain insight into the origin of the specificity, the binding to aspartyl-tRNA synthetase (AspRS) of the negatively charged substrate aspartic acid and the neutral analogue asparagine are compared by use of molecular dynamics and free energy simulations. Simulations of the Asn - AspRS complex show that although Asn cannot bind in the same position as Asp, several possible positions exist 1.5 to 2 A away from the Asp site. The binding free energy of Asn in three of these positions was compared to that of Asp through alchemical free energy simulations, in which Asp is gradually mutated ito Asn in the complex with the enzyme. To correctly account for the electrostatic interactions in the system (including bulk solvent), a recently developed hybrid approach was used, in which the region of the mutation site is treated microscopically, whereas distant protein and solvent are treated by continuum electrostatics. Seven free energy simulations were performed in the protein and two in solution. The various Asn positions and orientations sampled at the Asn endpoints of the protein simulations yielded very similar free energy differences. The calculated Asp → Asn free energy change is 79.8(±1.5) kcal/mol in solution and 95.1(±2.8) kcal/mol in the complex with the protein. Thus, the substrate Asp is predicted to bind much more strongly than Asn, with a binding free energy difference of 15.3 kcal/mol. This implies that erroneous binding of Asn by AspRS is highly improbable, and cannot account for any errors in the translation of the genetic code. Almost all of the protein contributions to the Asp versus Asn binding free energy difference arise from an arginine and a lysine residue that hydrogen bond to the substrate carboxylate group and an Asp and a Glu that hydrogen bond to these; all four amino acid residues are completely conserved in AspRSs. The protein effectively “solvates” the Asp side-chain more strongly than water does. The simulations are analyzed to determine the interactions that Asn is able to make in the binding pocket, and which sequence differences between AspRS and the highly homologous AsnRS are important for modifying the amino acid specificity. A double or triple mutation of AspRS that could make it specific for Asn is proposed, and supported by preliminary simulations of a mutant complex.

Peptide synthetases are large enzymatic complexes that catalyze the synthesis of biologically active peptides in microorganisms and fungi and typically have an unusual structure and sequence. Peptide synthetases have recently been engineered to modify the substrate specificity to produce peptides of a new sequence. In this study we show that surfactin synthetase can also be modified by moving the carboxyl-terminal intrinsic thioesterase region to the end of the internal amino acid binding domains, thus generating strains that produce new truncated peptides of the predicted sequence. Omission of the thioesterase domain results in nonproducing strains, thus showing the essential role of this region and the possibility of obtaining peptides of different lengths by genetic engineering. Secretion of the peptides depends on the presence of a functional sfp gene.

System y+L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na+ replacement with K+ does not affect lysine transport, but markedly decreases the affinity of the transporter for L-leucine and L-glutamine. This observation suggests that the specificity of system y+L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na+, K+, Li+ and guanidinium ion. In agreement with the prediction, the specificity of system y+L was altered by the monovalent cations. In the presence of Na+, L-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - L-norleucine) strengthened binding. In contrast, bulkiness at the level of the beta carbon was detrimental. In K+, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li+ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na+; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards beta carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., L-leucine, L-glutamine, L-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations.

The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor–receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding motif of TNF-R55. This region has been previously recognized as a distinct functional domain that is both required and sufficient for the activation of neutral sphingomyelinase (N-SMase). Overexpression of full-length FAN enhanced N-SMase activity in TNF–treated cells, while truncated mutants of FAN produced dominant negative effects. The data suggest that FAN regulates ceramide production by N-SMase, which is a crucial step in TNF signaling.

Tryptophan auxotrophs of Escherichia coli in which mutations were mapped to the trpS locus (encoding tryptophanyl-tRNA synthetase) have been previously isolated. We have investigated the tryptophanyl-tRNA synthetase (TrpRS) purified from six auxotrophic strains for changes in amino acid activation and aminoacylation. Steady-state kinetic analyses show that these mutant TrpRS proteins have increases in the apparent KM for tryptophan, decreases in turnover number, or both, without significant changes in the apparent KM for ATP or tRNA(Trp). The crystal structure of a highly homologous tryptophanyl-tRNA synthetase from Bacillus stearothermophilus in a complex with the cognate aminoacyl adenylate allowed us to place the mutations in a structural context. The mutations in the enzymes are located in the KMSKS loop (M196I), in or near the active site (D112E, P129S, A133E) or far from the active site. The last three mutants (T60R, L91F, G329S) could not be predicted by examination of the protein structure as they line an interface between the C-terminal alpha-helix of one subunit and the Rossmann folds of both subunits, thus affecting a specific region of the dimer interface. These results support a role for dimerization in properly configuring the two active sites of the dimeric enzymes in the Trp/Tyr subclass of class I aminoacyl-tRNA synthetases in order to achieve optimal catalysis.

Fullerenol was used as sweet taste receptor model to investigate the binding affinities of structurally related pairs of enantiomers by isothermal titration calorimetry (ITC). It reveals that amino acid binding with fullerenol are enthalpy-cost and entropically-driven processes, whereas enthalpy contributes to monosaccharide binding to fullerenol. Spontaneous binding of amino acids was found through two sequential steps in which the sweeter enantiomer displays larger binding constants. Association of the d-form of fructose and l-form galactose with fullerenol suggested that, the higher the perceived sweetness intensity of the enantiomer, the larger was the binding constant with respect to their antipodes. Further investigation by molecular dynamic simulation showed that the binding energy and the perceived sweetness intensity were well correlated. The preliminary results of this biomimetic research cover the lack of information about the thermodynamic basis of sweet sensation and the underlying principles of sweetness differences between the enantiomers of amino acids and monosaccharides.

Background: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II aminoacyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. Results The crystal structure of E. coli LysU has been determined to 2.8 a resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. Conclusion A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase–ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

The constitutive lysyl-tRNA synthetase (LysRS) of the Escherichia coli strain OEL134 differs from the wild-type enzyme by the single substitution of threonine 208 with methionine. In vitro study of the isotopic [32P]PPi-ATP exchange reaction catalyzed by purified T208M LysRS revealed specific features that are not observed with the wild-type LysRS: (i) The steady state of the reaction was reached after a approximately 1-min lag when the addition of the enzyme was used to initiate the reaction. This lag disappeared upon preincubation of the enzyme with lysine and ATP. (ii) The variation of the steady state rate as a function of the lysine concentration in the assay was sigmoidal (Hill coefficient of 1.65), suggesting cooperativity of lysine binding to this dimeric enzyme. The allosteric behavior of the mutant enzyme was further established by showing that, at low concentrations of lysine, low amounts of cadaverine stimulated T208M LysRS activity. T208A LysRS, in which threonine 208 had been changed into alanine by site-directed mutagenesis, displayed the same properties as T208M LysRS. Remarkably, Thr 208 makes part of the first signature motif of class II aminoacyl-tRNA synthetases, a motif likely to be involved in the dimerization of the enzyme subunits. Therefore, the behavior of the Thr 208 mutants of LysRS supports the idea that the dimerization of class II aminoacyl-tRNA synthetases is important for an efficient structuration of their active site.

We report large scale molecular dynamics computer simulations, ∼100 ns, of the ion pump Ca(2+)-ATPase immersed in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The structure simulated here, E1, one of the several conformations resolved using X-ray diffraction techniques, hosts two Ca(2+)-ions in the hydrophobic domain. Our results indicate that protonated residues lead to stronger ion-residue interactions, supporting previous conclusions regarding the sensitivity of the Ca(2+) behaviour to the protonated state of the amino acid binding sites. We also investigate how the protein perturbs the bilayer structure. We show that the POPC bilayer is ∼12% thinner than the pure bilayer, near the protein surface. This perturbation decays exponentially with the distance from the protein with a characteristic decay length of 0.8 nm. We find that the projected area per lipid also decreases near the protein. Using an analytical model we show that this change in the area is only apparent and it can be explained by considering the local curvature of the membrane. Our results indicate that the real area per lipid near the protein is not significantly modified with respect to the pure bilayer result. Further our results indicate that the local deformation of the membrane around the protein might be compatible with the enhanced protein activity observed in experiments over a narrow range of membrane thicknesses.

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using single cell imaging. Interestingly, mutating to different residues at two locations near the hinge region of the ECD could lead to either significantly lose of function of the receptor or gain of function (switch function mutations). Amino acid binding results in differential rescue effect in altering intracellular calcium responses, especially calcium oscillations. We further analyzed the effect of mutation and amino acid binding on the correlation motion, cooperativity, and synergistic activation use computational methods. These results provide important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--an inorganic divalent cation, and another hand, a nutrient-- how the receptor senses these agonists in healthy and diseased states.

The purpose of the following experiments was to describe some of the neurochemical changes that occur in the basal ganglia of rats exposed chronically to a classical neuroleptic, fluphenazine, and to relate these changes to extrapyramidal motor dysfunction. For these studies a combination of behavioural, receptor autoradiographic and in situ hybridization methods were employed. Preliminary pharmacological studies on GABA receptors showed that incubation in Tris-acetate rather than Tris-citrate buffer increased the number of binding sites labelled by [ 3 H]muscimol by over 120% without affecting binding affinity or selectivity. The results of experiments with fluphenazine showed that treatment for six months increased the frequency of vacuous chewing movements compared to controls. In the striatum, changes in GABA transmission were observed in fluphenazine-treated rats with increases in glutamate decar☐ylase mRNA levels in the caudate nucleus, dorsal shell and core of the accumbens and decreases in [ 3 H]muscimol binding in the caudate and dorsal shell regions. These data suggest that fluphenazine treatment increased GABA transmission in specific subregions of the candate and accumbens nuclei. In addition, glutamate decar☐ylase mRNA levels were elevated in the entopeduncular nucleus of fluphenazine-treated animals. Autoradiographic analysis of excitatory amino acid binding showed that fluphenazine exposure decreased [ 3 H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding in entopeduncular nucleus and in the ventrolateral thalamic nucleus and decreased [ 3 H]dizocilpine maleate binding in the medial geniculate nucleus. These experiments show that in addition to altering GABA transmission, chronic neuroleptic exposure alters excitatory amino acid transmission in specific regions of the basal ganglia-thalamocortical motor system. The neuroleptic dependent increases in glutamate decar☐ylase mRNA levels in the entopeduncular nucleus may reflect changes in neurotransmission in the indirect pathway connecting the major input and output nuclei of the basal ganglia. Changes in some of these brain regions may be related to the occurrence of extrapyramidal motor disturbances.

By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.

Background Genetic variation at human leukocyte antigen (HLA) alleles influences many phenotypes and mediate susceptibility to a wide range of human diseases include Immunoglobulin A Deficiency (IgAD). The high resolution SNP genotyping has enabled large cohort studies in this complex region. Aim To Investigate the HLA association of IgAD using high resolution SNP data and to identify the combinations of biologically relevant amino acid (AA) residues directly involved in disease. Methods HLA imputation was performed in 1046 samples including 758 IgAD individual and 288 ethnically matched controls. The imputation results were further confirmed by high resolution typing. Subsequently, multiple tools include SKDM HLA tools, SNP2HLA and Plink were used to investigate the amino acid binding pocket association in IgAD cohorts. Finally, 3D visualization of the predicted HLA amino acid sequence was performed using RaptorX. Results Strong IgAD association were observed in HLA-B*0801 ( p = 5.364 ∗ 10 −14 ), HLA-B*1402 ( p = 0.0016), HLA-DRB1*0301 ( p = 3.276 ∗ 10 −12 ), HLA-DRB1*0102 ( p = 0.001 ∗ 10 −12 ), HLA-DRB1*1201 ( p = 1.684 ∗ 10 −4 ), HLA-DQB1*0201 ( p = 1.835 ∗ 10 −12 ). Conversely, HLA-B*1501 ( p = 1.358 ∗ 10 −5 ), HLA-B*0702 ( p = 5.662 ∗ 10 −7 ), HLA-DRB1*1501 ( p = 6.34 ∗ 10 −23 ), HLA-DQB1*0402 ( p = 0.004) and HLA-DQB1*0602 ( p = 9.396 ∗ 10 −23 ) showed strong protection against IgAD. In addition, most significant AA residues in different HLA region associated with IgAD are: HLA-B ((AA 9, Asp, pockets 2 and 3, p = 8.047 ∗ 10 −14 ); (AA 156, Asp, pockets 4 and 5, p = 4.396 ∗ 10 −13 )); HLA-DRB1 ((AA 26, Tyr, pocket 4, p = 8.249 ∗ 10 −10 ); (AA 67, Leu, pocket 7, p = 5.236 ∗ 10 −8 )); HLA-DQB1 ((AA 38, Val, pocket 9, p = 4.894 ∗ 10 −10 ); (AA 37, Ile, pocket 9, p = 8.216 ∗ 10 −9 ), (AA 28 Ser and AA 47 Phe, pocket 7, p = 8.216 ∗ 10 −9 ); (AA 30, Ser, pocket 6, p = 8.216 ∗ 10 −9 ); (AA 71, Lys, pockets 4 and 7, p = 8.216 ∗ 10 −9 )). Conclusion The results suggested that certain peptide-binding residues of the HLA class I and class II molecule is associated with susceptibility to IgAD.

Neurotrophic factors, in particular NGF, have been shown to potently protect against glutamate-receptor-mediated toxicity. In order to further investigate the mechanism of this protection, we investigated the in vivo effects of fibroblasts, genetically modified to secrete NGF and implanted near the striatum, on striatal excitatory amino acid binding and receptor expression, on the induction of the peroxidative enzyme catalase, and on cellular energy metabolism in the striatum. Seven days after implantation into the corpus callosum of either a genetically altered NGF-producing (NGF[+]) or unaltered parental (NGF[-]) fibroblast cell-line, there is a time point at which NGF[+] cells have been shown to prevent exitotoxic insults. At that time point after implantation, we found that NGF[+] grafts caused a marked increase in catalase mRNA expression in and around the NGF[+] grafts. The NGF[+] grafts also reduced basal levels of striatal ATP when compared to the effects of NGF[-] grafts. No changes were observed in [ 3 H]glutamate binding and NMDA receptor mRNA expression. We conclude that effects of NGF[+] fibroblast grafts on glutamate receptor mediated toxicity are not by direct effects on glutamate receptors or glutamate binding, but rather appear to be a process involving enzymatic induction and modification of cellular energy stores. The observed increase in catalase mRNA suggests that peroxidative metabolism may be involved in these NGF-mediated effects.

Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity. To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases. We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size. Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine. The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size. For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated. Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E. coli phenylalanyl-tRNA synthetase.

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

L-Phenylalanine dehydrogenase catalyzes the NAD(+)-dependent, reversible, oxidative deamination of L-phenylalanine to form ammonia, phenyl pyruvate, and NADH. The enzyme has been purified to homogeneity from Rhodococcus sp. M4, and a partial amino acid sequence was obtained. A cosmid library of Rhodococcus sp. M4 genomic DNA was prepared and used to isolate a 2.5-kilobase PstI fragment that contained the pdh gene. The open reading frame of 1068 nucleotides encodes a polypeptide of 356 amino acids, portions of which match the amino acid sequence determined for the purified enzyme. Expression of the Rhodococcus pdh gene in Escherichia coli, which does not contain a phenylalanine dehydrogenase activity, yields a soluble enzyme exhibiting phenylalanine dehydrogenase activity. Both the enzyme purified from Rhodococcus and the enzyme expressed in E. coli are post-translationally modified by removal of the amino-terminal methionine. The overall amino acid sequence is homologous to previously reported sequences of leucine and phenylalanine dehydrogenases as well as several glutamate dehydrogenases. The amino-terminal portion of the enzyme contains residues involved in L-amino acid binding and catalysis, while the carboxyl-terminal portion contains the presumptive dinucleotide-binding domain. A detailed sequence comparison of Rhodococcus phenylalanine dehydrogenase with leucine, phenylalanine, and glutamate dehydrogenases suggests residues involved in general amino acid binding and others that provide for amino acid discrimination.

Alchemical molecular dynamics simulations are performed to determine the difference in the free energy of binding of the tyrosine substrate between the wild type of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and the mutant Tyr169-->Phe. The results are of general interest because the Tyr169 hydroxyl group interacts with the ammonium group of the substrate in a manner corresponding to that found in other amino acid binding proteins (e.g. the Asp receptor of the chemotactic bacterium Salmonella typhimurium and class I major histocompatibility complex molecules). The calculated free-energy change due to the Tyr169-->Phe mutation is 3.4 kcal/mol (the statistical error is +/- 0.5 kcal/mol) in satisfactory agreement with the experimental value of 3(+/- 0.5) kcal/mol. By use of thermodynamic integration, the contribution of the different terms to the free energy change are estimated. The path dependence of such a decomposition is discussed and it is suggested that the alchemical choice is of primary interest for understanding the interactions involved. There are large protein contributions to the alchemical free energy difference of the bound and free enzyme that cancel in the overall result. Due to this cancellation, the essential interactions contributing to the free-energy change are those between the OH group of Tyr169 and water in the free enzyme and those between the OH group of Tyr169 and the ammonium group of the substrate in the bound system. The results thus support simple models based on a balance of hydrogen bonding interactions.

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M . galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate l -arginine (88.22%), compared to l -alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or β-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M . galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate ( P . maximus ) .

Background. Reports of positive patch test and photopatch test reactions to the chemical ultraviolet filter octocrylene have increased during the last decade. Little is known about the reason for octocrylene's allergenic activity. Objectives. To present and discuss the results of patch tests and photopatch tests with octocrylene, and to investigate the possible cause of its allergenic properties. Methods. Results of patch tests and photopatch tests with octocrylene in patients with adverse skin reactions to sunscreen products and/or ketoprofen were collected. The allergenic potency of octocrylene was investigated in the murine local lymph node assay (LLNA). Chemical reactivity assays were used to mimic octocrylene's interaction with biomolecules. Results. We report 23 cases of positive test reactions to octocrylene (5 patch test and 18 photopatch). Notably, many of these patients also had positive photopatch test reactions to ketoprofen and benzophenone-3. Octocrylene was shown to be a moderate sensitizer in the LLNA, and it reacted with amines such as lysine, but not with thiols such as cysteine. Conclusions. The clinical studies show that octocrylene is both a photocontact allergen and a contact allergen. Octocrylene's ability to cause contact allergy is probably attributable to its reactivity towards lysine. To be able to understand why octocrylene causes photocontact allergy, further studies are needed.

We have analysed the sequence homology between glutamate, leucine and phenylalanine dehydrogenases in the light of the solution of the structure of the glutamate dehydrogenase from Clostridium symbiosum. This analysis indicates that the elements of secondary structure comprising the core of the two domains in glutamate dehydrogenase are conserved in the other two enzymes. There is a striking conservation of the residues responsible for the recognition of the nicotinamide ring of the nucleotide cofactor and the backbone of the amino acid substrates. Furthermore, residues involved in a major conformational rearrangement on amino acid binding are preserved, as are those implicated in the catalytic chemistry. In contrast, the pattern of insertions/deletions between these enzymes is consistent with possible differences in quaternary structure. Differential substrate specificity between these enzymes is achieved by critical substitutions at the base of the binding pocket, which accommodates the side-chain of the amino acid substrate. This provides insights into the mutations necessary to produce new catalysts for the chiral synthesis of novel amino acids.