Protein and RNA dynamics play key roles in determining the specific recognition of GU-rich polyadenylation regulatory elements by human Cstf-64 protein.

The N-terminal domain of the 64 kDa subunit of the cleavage stimulation factor (CstF-64) recognizes GU-rich elements within the 3'-untranslated region of eukaryotic mRNAs. This interaction is essential for mRNA 3' end processing and transcription termination, and its strength affects the efficiency of utilization of different polyadenylation sites. The structure of the RNA-binding N-terminal domain of CstF-64 showed how the N-terminal RNA recognition motif of CstF-64 recognizes GU-rich RNAs. However, it is still perplexing how this protein can bind selectively to RNAs that are rich in G and U residues regardless of their detailed sequence composition, yet discriminate effectively against non-GU-RNAs. We investigated by NMR the dynamics of the CstF-64 RNA-binding domain, both free and bound to two GU-rich RNA sequences that represent polyadenylation regulatory elements. While the free protein displays the motional properties typical of a well-folded protein domain and is uniformly rigid, the protein-RNA interface acquires significant mobility on the micro- to millisecond time-scale once GU-rich RNAs binds to it. These motional features, we propose, are intrinsic to the functional requirement to bind all GU-rich sequences and yet to discriminate against non-GU-rich RNAs. This behavior may be a general mechanism by which some RNA-binding proteins are able to bind to classes of sequences, as opposed to a well-defined sequence or consensus.

Solution structure of Archaeglobus fulgidis peptidyl-tRNA hydrolase (Pth2) provides evidence for an extensive conserved family of Pth2 enzymes in archea, bacteria, and eukaryotes.

The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6-kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four alpha-helices and a mixed beta-sheet consisting of four parallel and anti-parallel beta-strands, where the alpha-helices sandwich the beta-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii, and Sulfolobus solfataricus reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Data Bank but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good-quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal, and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli.

Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E.coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.

Heparin binding to cobra basic phospholipase A2 depends on heparin chain length and amino acid specificity.

Heparin is shown to bind specifically to the carboxy-terminal region of toxic type I phospholipase A2 from Naja nigricollis (N-PLA2) by competition assay using synthetic polypeptides and heparin affinity chromatography. The binding strength is seen to depend on heparin chain length and the presence of N-sulfate groups of heparin. It is observed that both electrostatic and non-electrostatic interactions are involved in the specific binding of heparin to the carboxy-terminus. When heparin's size is at least a decasaccharide, about two molecules of N-PLA2 bind to one molecule of heparin, as evidenced by the chemical estimate of protein to carbohydrate ratio in such N-PLA2/heparin complexes. Based on such a stoichiometric measurement and computer modeling of the N-PLA2/heparin complex, it is suggested that the binding sites of the two N-PLA2 molecules on one heparin molecule lie on the opposite sides of the heparin chain.

Trypanothione as a target in the design of antitrypanosomal and antileishmanial agents.

Trypanothione is the key molecule in the defence mechanism of Trypanosoma and Leishmania against oxidative stress. The uniqueness of trypanothione makes the metabolism of this molecule an attractive target in antitrypanosomal and antileishmanial drug design. It became clear that this antioxidant cascade can be considered as the "Achilles heel" of these parasites. The following targets and their respective inhibitors will be discussed: biosynthesis of trypanothione with glutathionylspermidine synthetase and trypanothione synthetase; biosynthesis of glutathione with gamma-glutamylcysteine synthetase; biosynthesis of spermidine with ornithine decarboxylase; trypanothione hydroperoxide metabolism with tryparedoxine peroxidase, tryparedoxine and trypanothione reductase.

Characterization of the peptide substrate specificity of glutathionylspermidine synthetase from Crithidia fasciculata.

Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (gamma-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the gamma-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine with methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution of L-Cys by amino acids with an alkyl side-chain is also well tolerated giving relative catalytic efficiencies of 1.1 and 1.5 for peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.

Optimal real correlation filters.

Expressions are derived for real filters that have a maximum correlation signal to noise ratio. Both continuous and discrete cases are treated and shown to have similar forms. The signal can be complex, and the case of a real signal is considered and related to previous results.

Action of Taiwan cobra cardiotoxin on membranes: binding modes of a beta-sheet polypeptide with phosphatidylcholine bilayers.

The interaction of Taiwan cobra cardiotoxin (CTX A3), a basic polypeptide consisting of three-fingered loops and five-strand beta-sheet structure, with zwitterionic dipalmitoylphosphatidylcholine (DPPC) has been studied by 31P and 2H NMR to understand the binding modes of CTX in membrane bilayers. The results, in conjunction with DPH fluorescence anisotropy and differential scanning calorimetry studies, show that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple Pbeta' phase. Elevating the temperature to that of the liquid crystalline Lalpha phase leads to the fusion of the small aggregates into larger ones as evidenced by the change of the isotropic signal into a magnetically aligned 31P signal with a marked reduction in the chemical shift anisotropy. 2H NMR study on deuterium-labeled DPPC in the head group and fatty acyl region as a function of temperature and CTX concentration reveals a molecular model that CTX undergoes a redistribution between penetrating and peripheral binding states depending on the temperature studied. In addition, both the conformational and dynamic states of the phosphocholine head group of DPPC bilayers are significantly perturbed in the presence of CTX. Structural consideration of the CTX molecule indicates that the penetration binding mode of CTX with the DPPC bilayer may involve a novel membrane-binding motif identified recently in the three-fingered loops of P-type CTX. CTX can only bind to DPPC membrane peripherally in the Lalpha phase due to the mismatch of their hydrophobic lengths.