Stabilizing the eIF4G1 α-helix increases its binding affinity with eIF4E: implications for peptidomimetic design strategies.

Eukaryotic initiation factor (eIF)4E is overexpressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins such as c-Myc, cyclin D1, and vascular endothelial growth factor. Inhibition of eIF4E is therefore a potential therapeutic target for human cancers. A novel peptide based on the eIF4E-binding peptide eIF4G1, where the α-helix was stabilized by the inclusion of α-helix inducers as shown by CD measurements, was synthesized. The helically stabilized peptide binds with an apparent K(d) of 9.43±2.57 nM, which is ∼15.7-fold more potent than the template peptide from which it is designed. The helically stabilized peptide showed significant biological activity at a concentration of 400 µM, unlike the naturally occurring eIFG1 peptide when measured in cell-based cap-dependent translational reporter and WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assays. Fusion of the template peptide and the stabilized peptide to the cell-penetrating peptide TAT produced more active but equally potent inhibitors of cap-dependent translation in cell lines. They also equally disrupted cell metabolism as measured in a WST-1 assay. Propidium iodide staining revealed that the TAT-fused, helically stabilized peptide caused more cell death than the TAT-fused eIF4G1 template peptide with substantial decreases in the G1 and G2 cell populations. Annexin-staining experiments also indicated that the TAT-fused eIF4G1 derivative peptides caused cell death by apoptosis. The results presented should offer further insight into peptidomimetics development for eIF4E.

Updates on p53: modulation of p53 degradation as a therapeutic approach.

The p53 pathway is aberrant in most human tumours with over 50% expressing mutant p53 proteins. The pathway is critically controlled by protein degradation. Here, we discuss the latest developments in the search for small molecules that can modulate p53 pathway protein stability and restore p53 activity for cancer therapy.

Protein flexibility: its role in structure and mechanism revealed by molecular simulations.

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques.

Rational design of thermally stable proteins: relevance to bionanotechnology.

Design of thermally stable proteins is spurred by their applications in bionanotechnology. There are three major issues governing this: first, the upper limit on the temperature at which proteins remain physiologically active and are available for technological applications (answers may emerge from the discovery of new, natural hyperthermophilic enzymes that are active above 125 degrees C or from the selection of mutants of hyperthermophilic enzymes that are more stable); second, the use of hyperthermophilic enzymes as molecular templates to design highly stable enzymes that have high activity at low temperatures; third, the link between rigidity and flexibility to thermostability and activity, respectively. We review progress in these areas.

Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.

The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically.

Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis.

We have built a homology model of the AAA domain of the ATP-dependent protease FtsH of Escherichia coli based on the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. The resulting model of the hexameric ring of the ATP-bound form of the AAA ATPase suggests a plausible mechanism of ATP binding and hydrolysis, in which invariant residues of Walker motifs A and B and the second region of homology, characteristic of the AAA ATPases, play key roles. The importance of these invariant residues was confirmed by site-directed mutagenesis. Further modelling suggested a mechanism by which ATP hydrolysis alters the conformation of the loop forming the central hole of the hexameric ring. It is proposed that unfolded polypeptides are translocated through the central hole into the protease chamber upon cycles of ATP hydrolysis. Degradation of polypeptides by FtsH is tightly coupled to ATP hydrolysis, whereas ATP binding alone is sufficient to support the degradation of short peptides. Furthermore, comparative structural analysis of FtsH and a related ATPase, HslU, reveals interesting similarities and differences in mechanism.

Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis.

The small, DNA-binding protein GerE regulates gene transcription in the terminally differentiated mother-cell compartment during late stages of sporulation in Bacillus subtilis. This versatile transcription factor shares sequence homology with the LuxR/FixJ/UhpA family of activators and modulates the expression of a number of genes, in particular those encoding the components of the coat that surrounds the mature spore. GerE orchestrates the final stages of coat deposition and maturation that lead to a spore with remarkable resistance properties but that must be responsive to low levels of germination signals. As this germination process is largely passive and can occur in the absence of de novo protein synthesis, the correct assembly of germination machinery, including germinant receptors and energy storage compounds, is crucial to the survival of the cell. The crystal structure of GerE has been solved at 2.05 A resolution using multi-wavelength anomalous dispersion techniques and reveals the nature of the GerE dimer. Each monomer comprises four alpha-helices, of which the central pair forms a helix-turn-helix DNA-binding motif. Implications for DNA-binding and the structural organisation of the LuxR/FixJ/UhpA family of transcription activator domains are discussed.

Understanding the mechanism of ice binding by type III antifreeze proteins.

Type III antifreeze proteins (AFPs) are present in the body fluids of some polar fishes where they inhibit ice growth at subzero temperatures. Previous studies of the structure of type III AFP by NMR and X-ray identified a remarkably flat surface on the protein containing amino acids that were demonstrated to be important for interaction with ice by mutational studies. It was proposed that this protein surface binds onto the (1 0 [\bar 1] 0) plane of ice with the key amino acids interacting directly with the water molecules in the ice crystal. Here, we show that the mechanism of type III AFP interaction with ice crystals is more complex than that proposed previously. We report a high-resolution X-ray structure of type III AFP refined at 1.15 A resolution with individual anisotropic temperature factors. We report the results of ice-etching experiments that show a broad surface coverage, suggesting that type III AFP binds to a set of planes that are parallel with or inclined at a small angle to the crystallographic c-axis of the ice crystal. Our modelling studies, performed with the refined structure, confirm that type III AFP can make energetically favourable interactions with several ice surfaces.

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed.