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PubMed2003

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein.

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.

Amino Acid Substitution↗

PubMed1982

Kinetics of acyl transfer ribonucleic acid complexes of Escherichia coli phenylalanyl-tRNA synthetase. A conformational change is rate limiting in catalysis.

Kinetics of complex formation between phenylalanyl-tRNA synthetase and Phe-tRNAPhe have been measured by the stopped-flow technique. Either the protein intrinsic fluorescence or the fluorescence of the added indicator 6-(p-toluidinyl)naphthalene-2-sulfonate was observed. Identical results were obtained with each method. Acyl-tRNAs with variable structures of the acyl and tRNA moieties were examined. Kinetics were measured as a function of pH and at different ionic strengths. Kinetic constants were compared with those of enzymatic phenylalanylation of tRNAPhe. The results are as follows. (1) Phe-tRNAPhe binds to phenylalanyl-tRNA synthetase in two, mutually exclusive types of complexes, one at the tRNA-specific binding site of the enzyme and the other in a region which involves the Phe-specific binding site of the enzyme [Holler, E. (1980) Biochemistry 19, 1397-1402]. The Phe site directed association includes a conformational change of the complex that is rate limiting. (2) The conformational change and catalytic tRNA aminoacylation follow similar values of rate constants irrespectively of pH and ionic strength. It is concluded that aminoacylation is limited by the kinetics of a conformational change of the nascent enzyme-Phe-tRNAPhe complex. (3) The nature of Phe site directed binding was probed by variation of the structure of Phe-tRNAPhe. Both the acyl and the tRNA moieties are recognized by the enzyme. Of the acyl moiety, only the phenyl ring but not the amino group is essential for binding. The amino group can be acetylated or replaced by a hydroxyl group. Protonation of the amino group results in loss of Phe site directed binding. It gives a pKa = 6.9, which is close to that for protonation of a phenylalanine ester. (4) Rate constants were only slightly affected by addition of 200 mM NaCl at pH 7.5, indicating that the contribution by electrostatic forces was probably minimal. Mg2+ ions were essential for Phe site directed binding. Complexation of enzyme, Phe-tRNAPhe, and Mg2+ either was random or was at preequilibrium with the conformational change. (5) Binding of Phe-tRNAPhe at the tRNA-specific site of the enzyme was studied in the presence of in situ synthesized phenylalanyl adenylate. The reaction was bimolecular with rate constants of 50 microM-1 s-1 and 15 s-1 for association and dissociation, respectively.

Amino Acyl-tRNA Synthetases↗

PubMed2007

Evolutionarily conserved structural and functional roles of the FYVE domain.

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Amino Acid Motifs↗

PubMed1991

Eukaryotic DNAs in solution contain characteristic components of tertiary structure.

For natural eukaryotic DNA in solution, we suggest the existence of secondary-helix components superponed to parts of the DNA double helices. In a previous report we found, for calf thymus DNA in solution and of different mean molar mass Mr, an electrostatically driven rise of the hydrodynamically operative contour length of the double helix. This result was derived from Mr-dependent systematic deviations from the almost but not exactly linear plots of intrinsic viscosity [eta] as a function of 1/cs1/2 (cs = Na+ concentration) accurately determined by a titration technique [K.G. and K.E.R., Nucl. Acids Res. 8, 2807 (1980)]. In order to discriminate between DNA elongation contributions caused by secondary or by tertiary structure effects, respective measurements have now been extended to different temperatures for two eukaryotic and two prokaryotic DNA species. The slope of the curves obtained for the (apparent) gradual elongation effect as a function of temperature is negative for the eukaryotic DNAs investigated and is smaller and positive for the prokaryotic species, thus revealing different underlying main elongation mechanisms. We propose that, for the eukaryotic DNA samples, an electrostatically driven partial abolition of tertiary structure components is responsible for the prevailing part of the DNA elongation effect measured. (A helix elongation of this type may be the result of an abolition of an apparent helix shortening as realized in a very high degree on formation of nucleosome chains or in a less degree by DNA molecules with a respective evolutionarily fitted tertiary structure). For the smaller effects of prokaryotic DNA species something like a base breathing seems to dominate. Recent literature results support such an interpretation.

Animals↗

PubMed2003

Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.

The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.

Amino Acid Sequence↗

PubMed1991

Structure and function of ribonuclease A binding subsites.

Ribonuclease A binds nucleic acids through multiple electrostatic interactions between the phosphates of the polynucleotide and the positive groups (side chains of lysines and arginines) of the protein subsites. The bases only play a significant role in the binding at the active site. The active centre p1R1B1 sites determine the specificity of the catalytic cleavage. The phosphate-binding subsites p2 (Lys-7 and Arg-10), p1 (Lys-41, His-12 and His-119) and p0 (Lys-66) are essential for an effective catalysis and are conserved in all mammalian pancreatic ribonucleases. Additional phosphate-binding subsites confer further catalytic efficiency, probably by avoiding non-productive binding. The minimum chain size for optimum catalysis is probably longer than six or seven nucleotides. The full occupancy of binding sites by the long chain polynucleotides would explain the preference of the enzyme for these substrates. The multiplicity of binding subsites is responsible for the helix-destabilizing activity of ribonuclease A. Its capacity for destroying the secondary structure of single-stranded nucleic acids may be of importance for the complete hydrolysis of RNA in the digestive tract. A large variety of proteins, with very different structures and functions, interact with nucleic acids. An analysis of their binding properties shows that there is no general model for protein-nucleic acid interaction. However, the vast amount of work on the ribonuclease A binding subsites should serve as a model for the study of the binding properties of many other proteins that recognize nucleic acids.

Animals↗

PubMedDate not supplied

[Correlation between electrokinetic parameters of isolated hepatocyte nuclei and their functional activity in rats].

The results of investigations of the electrokinetic properties of cell nuclei in various functional states were summarized. A positive correlation between the value of the electrostatic charge on the nuclear membrane surface and the functional state of the genetic apparatus upon activation was established. It was shown that the value of the electrokinetic potential reflects differences in the mechanisms of action of biologically active substances, and that this may be related to the peculiarity of their action on both membrane surface and the cell genome. It is concluded that the electrokinetic properties of the nuclear membrane reflect changes occurring in the genome of the eukaryotic cell nucleus, and from the value of surface charge density, the extent of involvement of the genome under various influences can be determined.

Amino Acids↗

PubMed1985

High-performance liquid chromatography of tRNAs on novel stationary phases.

Rapid separation of a group of tRNAs was carried out on novel siliceous bonded stationary phases with aqueous eluents by using gradient elution with increasing or decreasing salt gradient, as usual in electrostatic interaction chromatography or hydrophobic interaction chromatography, respectively. The stationary phases consist of microparticulate macroporous silica with surface-bound polar moieties, containing weak cationic and/or hydrophobic binding sites. Depending on the nature of the binding sites, the stationary phases exhibit different retention behavior and selectivity for tRNAs. Aqueous phosphate solutions were used as the eluent, and in many cases isocratic elution was sufficient to separate seven tRNAs. Addition of magnesium ions or n-decylbetaine to the eluent resulted in lower retention, the latter causing a greater increase in the eluent strength. The optimum pH range of the eluent was 5.5-6.5.

Betaine↗

PubMed2003

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase.

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.

Amino Acid Motifs↗

PubMed1997

The role of Glu73 of barnase in catalysis and the binding of barstar.

Barnase, a small extracellular ribonuclease from Bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly. Barnase has also evolved to be catalytically active. The active site of barnase and its binding site for barstar use the same subset of amino acids. The exception is Glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar. We examined in this work the contribution of Glu73 to both catalysis and barstar binding. Truncation mutants of the general base (Glu73 --> Ala or Ser) retain a residual RNase activity of about 0.3% while mutants with larger hydrophobic replacements (Glu 73 --> Trp or Phe) have virtually no catalytic activity. This, and binding data of 3'-GMP with the different barnase mutants suggest that the loss in activity results from the elimination of the general base, which can be substituted to some extent by water or other polar side-chains in truncation mutants. All of the Glu73 mutations lead to a weakening of the free energy of complex formation with barstar by 1.4 to 3.0 kcal/mol (including Gln). This is surprising, since Glu73 does not interact directly with barstar and there is an electrostatic repulsion between Glu73 on barnase and the negatively charged binding surface of barstar. A newly developed method of constructing double mutant cycles between multiple mutations at the same site appears to pinpoint a favourable interaction between Glu73 and one of its nearest neighbours in barstar, Asp39. The coupling energy between those residues is presumably indirect: the carboxylate of Glu73 organizes neighbouring positively charged groups in barnase, Lys27, Arg83, and Arg87 to interact with Asp39 in barstar. This emphasizes that an apparent interaction between a pair of residues as measured with double mutant cycles is the sum of their direct and indirect interactions.

Bacterial Proteins↗

PubMed1993

Theoretical analyses on the role of Mg2+ ions in ribozyme reactions.

To elucidate the role of the Mg2+ ion in ribozyme reactions, we carried out ab initio molecular orbital investigations on dianionic trimethoxyphosphorane A and its Mg2+ complex (overall a neutral molecule) as a model system for the reaction center of Tetrahymena-type ribozyme. Although dianionic oxyphosphorane A concentrates its negative charges on the equatorial phosphoryl oxygens, the coordination of the Mg2+ ion between these two oxygens is unlikely. Geometry optimizations of the complex and the electrostatic potential of A both suggest that Mg2+ coordination preferably occurs in the region between the axial oxygen and the equatorial phosphoryl oxygen. The considerations of electrostatic potential rationalize the geometries of carboxylate-metal and phosphate-metal interactions extracted from the Cambridge Structural Database as well. Consequently, the Mg2+ ion at the active site of Tetrahymena-type ribozyme most likely lies in the regions between the axial and equatorial oxygens. The axial-equatorial coordinations of Mg2+ ions conceivably increase the electronegativities of the axial oxygens and facilitate cleavage of the phosphodiester bond located at the junction of the intron and the exon. It is thus likely that the Mg2+ ions play the key role in the phosphodiester cleavage reactions mediated by ribozymes.

Animals↗

PubMed2003

Solvation change and ion release during aminoacylation by aminoacyl-tRNA synthetases.

Discrimination between cognate and non-cognate tRNAs by aminoacyl-tRNA synthetases occurs at several steps of the aminoacylation pathway. We have measured changes of solvation and counter-ion distribution at various steps of the aminoacylation pathway of glutamyl- and glutaminyl-tRNA synthetases. The decrease in the association constant with increasing KCl concentration is relatively small for cognate tRNA binding when compared to known DNA-protein interactions. The electro-neutral nature of the tRNA binding domain may be largely responsible for this low ion release stoichiometry, suggesting that a relatively large electrostatic component of the DNA-protein interaction free energy may have evolved for other purposes, such as, target search. Little change in solvation upon tRNA binding is seen. Non-cognate tRNA binding actually increases with increasing KCl concentration indicating that charge repulsion may be a significant component of binding free energy. Thus, electrostatic interactions may have been used to discriminate between cognate and non-cognate tRNAs in the binding step. The catalytic constant of glutaminyl-tRNA synthetase increases with increasing osmotic pressure indicating a water release of 8.4 +/- 1.4 mol/mol in the transition state, whereas little change is seen in the case of glutamyl-tRNA synthetase. We propose that the significant amount of water release in the transition state, in the case of glutaminyl-tRNA synthetase, is due to additional contact of the protein with the tRNA in the transition state.

Amino Acyl-tRNA Synthetases↗

PubMed2000

The solution structure of ribosomal protein L36 from Thermus thermophilus reveals a zinc-ribbon-like fold.

We have determined the solution NMR structure of the ribosomal protein L36 from Thermus thermophilus. L36 is the smallest protein in the large subunit of the prokaryotic ribosome. The sequence contains three completely conserved cysteine residues and one conserved histidine residue in a C-X(2)-C-X(12)-C-X(4)-H motif. Extended X-ray absorption fine structure spectroscopy was used to confirm that a purified L36 sample contains an equimolar amount of zinc. The structure of L36 was determined using simulated annealing based on NOE distance restraints, dihedral angle restraints and hydrogen bond distance restraints derived from NMR spectra of (15)N-labeled and non-labeled L36 samples at pH 7 and 12 degrees C, and by imposing tetrahedral zinc ion coordination geometry. The L36 fold is characterized by a triple-stranded antiparallel beta-sheet with the zinc-binding site at one end. The structure of the zinc site is well-determined and shows that the three cysteine sulphur atoms are supported by hydrogen bonds to backbone amide protons. The conserved histidine residue is located in a short 3(10)-helix and coordinates zinc by the N(delta1) atom. The electrostatic surface potential and location of conserved Arg, Lys and His side-chains suggest a large continuous L36-rRNA interaction interface. The folding topology as well as position and conformation of many conserved side-chains in L36 are very similar to those of zinc-ribbon domains found in the archaeal transcription factor TFIIB N terminus and the eukaryal transcription elongation factor hTFIIS C terminus. Given the relative antiquity of the ribosome it is possible that L36 reflects the parent of transcription-related zinc ribbons.

Amino Acid Motifs↗

PubMed2006

A charge-scaling implementation of the variational electrostatic projection method.

Two new charge-scaling methods for efficient modeling of the solvated macromolecular environment in hybrid QM/MM calculations of biological reactions are presented. The methods are extensions of the variational electrostatic projection (VEP) method, and allows a subset of atomic charges in the external environment to be adjusted to mimic, in the active dynamical region, the electrostatic potential and field due to the large surrounding macromolecule and solvent. The method has the advantages that it offers improved accuracy, does not require the use of a three-dimensional grid or auxiliary set of fitting points, and requires only minor molecular simulation code modifications. The VEP-cs and VEP-RVM+cs methods are able to attain very high accuracy (relative force errors of 10(-7) or better with appropriate choice of control parameters), and take advantage of a recently introduced set of high-order discretization schemes and Gaussian exponents for boundary element solvation and VEP methods. The methods developed here serve as potentially powerful tools in the arsenal of computational techniques used in multiscale computational modeling problems.

Biopolymers↗

PubMed2002

Crystal structure of human L-isoaspartyl methyltransferase.

The enzyme l-isoaspartyl methyltransferase initiates the repair of damaged proteins by recognizing and methylating isomerized and racemized aspartyl residues in aging proteins. The crystal structure of the human enzyme containing a bound S-adenosyl-l-homocysteine cofactor is reported here at a resolution of 2.1 A. A comparison of the human enzyme to homologs from two other species reveals several significant differences among otherwise similar structures. In all three structures, we find that three conserved charged residues are buried in the protein interior near the active site. Electrostatics calculations suggest that these buried charges might make significant contributions to the energetics of binding the charged S-adenosyl-l-methionine cofactor and to catalysis. We suggest a possible structural explanation for the observed differences in reactivity toward the structurally similar l-isoaspartyl and d-aspartyl residues in the human, archael, and eubacterial enzymes. Finally, the human structure reveals that the known genetic polymorphism at residue 119 (Val/Ile) maps to an exposed region away from the active site.

Binding Sites↗

PubMed1978

Interaction of aminoacridines with nucleic acids. A pulse-radiolysis study.

Pulse radiolysis has been used to study the interaction of aminoacridines with nucleic acids. The data confirm that there are two modes of binding. These are: a weak interaction which has a maximum binding ratio of one site per dye; and a strong binding process effected by both electrostatic and Van der Waals interactions. The limit of this latter, strong binding mode is approximately six sites per dye. The radiation-induced transient absorption spectrum of benzoflavine is characterized by a pronounced bleaching at 440 nm, which is quenched by the addition of nucleic acids. Mechanisms have been proposed for the reactions of both eaq-and .OH with benzoflavine which account both for the observed bleaching of benzoflavine solutions and for the protective effect of nucleic acids. It is proposed that eaq-reacts with benzoflavine to form a stable benzoflavine semiquinone radical and that .OH reacts with subsequent formation of a very stable benzoflavine hydroxycyclohexadienyl radical.

Acridines↗

PubMed2005

Identification of putative sulfurtransferase genes in the extremophilic Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins.

Eight nucleotide sequences containing a single rhodanese domain were found in the Acidithiobacillus ferrooxidans ATCC 23270 genome: p11, p14, p14.3, p15, p16, p16.2, p21, and p28. Amino acids sequence comparisons allowed us to identify the potentially catalytic Cys residues and other highly conserved rhodanese family features in all eight proteins. The genomic contexts of some of the rhodanese-like genes and the determination of their expression at the mRNA level by using macroarrays suggested their implication in sulfur oxidation and metabolism, formation of Fe-S clusters or detoxification mechanisms. Several of the putative rhodanese genes were successfully isolated, cloned and overexpressed in E. coli and their thiosulfate:cyanide sulfurtransferase (TST) and 3-mercaptopyruvate/cyanide sulfurtransferase (MST) activities were determined. Based on their sulfurtransferase activities and on structural comparisons of catalytic sites and electrostatic potentials between homology- modeled A. ferrooxidans rhodaneses and the reported crystal structures of E. coli GlpE (TST) and SseA (MST) proteins, two of the rhodanese-like proteins (P15 and P16.2) could clearly be defined as TSTs, and P14 and P16 could possibly correspond to MSTs. Nevertheless, several of the eight A. ferrooxidans rhodanese-like proteins may have some different functional activities yet to be discovered.

Acidithiobacillus↗

PubMed1994

Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis.

N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities. 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA. When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced. From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA. These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding). The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2. The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA. A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities.

Antineoplastic Agents↗