An intramolecular disulphide bond in human 4E-T affects its binding to eIF4E1a protein.

The cap at the 5'terminus of mRNA is a key determinant of gene expression in eukaryotic cells, which among others is required for cap dependent translation and protects mRNA from degradation. These properties of cap are mediated by several proteins. One of them is 4E-Transporter (4E-T), which plays an important role in translational repression, mRNA decay and P-bodies formation. 4E-T is also one of several proteins that interact with eukaryotic initiation factor 4E (eIF4E), a cap binding protein which is a key component of the translation initiation machinery. The molecular mechanisms underlying the interactions of these two proteins are crucial for mRNA processing. Studying the interactions between human eIF4E1a and the N-terminal fragment of 4E-T that possesses unstructured 4E-binding motifs under non-reducing conditions, we observed that 4E-T preferentially forms an intramolecular disulphide bond. This "disulphide loop" reduces affinity of 4E-T for eIF4E1a by about 300-fold. Considering that only human 4E-T possesses two cysteines located between the 4E binding motifs, we proposed that the disulphide bond may act as a switch to regulate interactions between the two proteins.

Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme.

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central ß-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer-monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

Hierarchical approach for the rational construction of helix-containing nanofibrils using α,ß-peptides.

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA Hp ) belongs to the well conserved family of serine proteases. HtrA Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrA Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrA Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Biochemical properties of the HtrA homolog from bacterium Stenotrophomonas maltophilia.

The HtrA proteins due to their proteolytic, and in many cases chaperone activity, efficiently counteract consequences of stressful conditions. In the environmental bacterium and nosocomial pathogen Stenotrophomonas maltophilia HtrA (HtrASm) is induced as a part of adaptive response to host temperature (37°C). We examined the biochemical properties of HtrASm and compared them with those of model HtrAEc from Escherichia coli. We found that HtrASm is a protease and chaperone that operates over a wide range of pH and is highly active at temperatures between 35 and 37°C. The temperature-sensitive activity corresponded well with the lower thermal stability of the protein and weaker stability of the oligomer. Interestingly, the enzyme shows slightly different substrate cleavage specificity when compared to other bacterial HtrAs. A computational model of the three-dimensional structure of HtrASm indicates differences in the S1 substrate specificity pocket and suggests weaker inter-trimer interactions when compared to HtrAEc. The observed features of HtrASm suggest that this protein may play a protective role under stressful conditions acting both as a protease and a chaperone. The optimal temperatures for the protein activity may reflect the evolutionary adaptation of S. maltophilia to life in soil or aqueous environments, where the temperatures are usually much below 37°C.

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans.

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB's importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates.

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy.

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5' cap.

Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E.

Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy­entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5'-5' triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution.

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin.

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m(7)Guo), and orthophosphate (P(i), natural cosubstrate) and its thiophosphate analogue (SP(i)), found to be a weak cosubstrate, have been studied in the pH range 5-8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK(a) 9.2 and 8.8); m(7)Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK(a) 7.0); and P(i) and SP(i) exhibit equilibria between monoanionic and dianionic forms (pK(a) 6.7 and 5.4, respectively). The phosphorolysis of m(7)Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP(i), independently of pH. With P(i), the human enzyme shows Michaelis kinetics only at pH approximately 5. However, in the pH range 5-8 for the bacterial enzyme, and 6-8 for the human enzyme, enzyme kinetics with P(i) are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P(i), characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K (m1) and K (m2)) and apparent maximal velocities (V (max1) and V (max2)). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K (m1) < K (m2)) and enzyme kinetics (V (max1)/K (m1) > V (max2)/K (m2)). Comparison of the pH-dependence of the substrate properties of P(i) versus SP(i) points to both monoanionic and dianionic forms of P(i) as substrates, with a marked preference for the dianionic species in the pH range 5-8, where the population of the P(i) dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n (H) approximately 1 to n (H) approximately 0.7 for Guo with the human enzyme, and to n (H) approximately 0.7 and 0.5 for m(7)Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP(i) in relation to its structure.