5-Substituted pyrimidines with a 1,5-anhydro-2, 3-dideoxy-D-arabino-hexitol moiety at N-1: synthesis, antiviral activity, conformational analysis, and interaction with viral thymidine kinase.

A new series of anhydrohexitol nucleosides are described. These compounds have a pyrimidine base moiety substituted in the 5-position with a chloro (1b), trifluoromethyl (1c), vinyl (1d), 2-thienyl (1e), ethynyl (1f) or propynyl (1g) substituent. The vinyl, propynyl, and, in particular, the 5-trifluoromethyl analogue showed potent activity against herpes simplex virus (HSV), 1c with a selectivity index of >16000 against HSV-1 and >1000 against HSV-2. Conformational analysis of anhydrohexitol nucleosides using computational methods indicates that these nucleosides occur in an equilibrium between the C1 and 1C form with a DeltaE of 5.9 kJ/mol. When the anhydrohexitol nucleoside is cocrystallized with the HSV-1 thymidine kinase it adopts a 1C conformation, which is opposite to the conformation found for the small molecule alone. The enzyme, apparently, induces a conformational change, and conformational flexibility of an anhydrohexitol nucleoside may be advantageous for recognition by viral enzymes.

Molecular characterization of a novel subtilisin inhibitor protein produced by Streptomyces venezuelae CBS762.70.

We report here on the isolation and identification of a gene coding for a novel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS762.70. The vsi gene was isolated on a 5-kb chromosomal PvuII fragment as identified by DNA sequencing and inhibitor activity testing of the gene product. Primer extension studies revealed that the mRNA transcriptional start point was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi coding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-terminal amino acid sequence analysis. Compared with the already known Streptomyces proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in VSI. As a consequence, VSI could be classified within a separate group of Streptomyces subtilisin inhibitors.

Some 6-aza-5-substituted-2'-deoxyuridines show potent and selective inhibition of herpes simplex virus type 1 thymidine kinase.

The synthesis and X-ray crystal structures of a series of 5-substituted-6-aza-2'-deoxyuridines is reported. These nucleoside analogues inhibit the phosphorylation of thymidine by HSV-1 TK but have no effect on the corresponding human enzyme. Detailed examination of one analogue proves it to be a competitive inhibitor of thymidine with a Ki of 0.34 microM and is a very poor substrate. The analogues are not substrates for the enzyme and also do not inhibit the degradation of thymidine by thymidine phosphorylase. Molecular modelling showed that the inhibitors fit well in the active site of HSV-1 TK, provided the conformation of the sugar moiety is the same for thymidine in the complex.

Calculation of pathways for the conformational transition between the GTP- and GDP-bound states of the Ha-ras-p21 protein: calculations with explicit solvent simulations and comparison with calculations in vacuum.

The transitions between the water-equilibrated structures of the GTP and GDP forms of Ha-ras-p21 have been calculated by using the targeted molecular dynamics (TMD) method (Schlitter et al., Mol. Sim. 10:291-309, 1993) both in vacuo and with explicit solvent simulation. These constrained molecular dynamics calculations result in different pathways, depending on the nucleotide bound. Each pathway consists in a sequence of transitions affecting six segments of the protein, four of them forming a hydrophilic cleft around the nucleotide. The transitions are initiated by the removal or introduction of the gamma-phosphate of the nucleotide and proceed sequentially, crossing several low-energy transition states. The movements are transmitted either by direct interactions between the segments or through the nucleotide. The GTP to GDP pathway is initiated by the removal of the nucleotide gamma-phosphate. This gives some space to Gly12, Gly13, and Val14. Their movement is transmitted to the target recognition domain and the switch II region, forcing these segments to adopt another position. In a second step the target recognition domain and the switch II region undergo conformational transitions to reach an intermediate conformation. Finally, there is a relaxation of the target recognition domain to its final state that forces the switch II region to reach its target conformation. The calculated pathways allow the identification of many residues that play an important role in the conformational changes, explain the altered transformation properties of some, and suggest mutations to alter the pathway.

Modelling pathways of alpha-chymotrypsin activation and deactivation.

The conformational change of alpha-chymotrypsin from an inactive, chymotrypsinogen like structure at high pH to an active conformation around pH 8.5 is used here as a model system to generate possible pathways for the transition by use of two different theoretical methods. One method, the 'targeted molecular dynamics' algorithm (TMD) adds a constraint in the direction of the target to a molecular dynamics force field and gives two different paths, one for every direction of the reaction (Schlitter,J., Engels,M., Krüger,P.J., Mol. Graphics (1994) 12, 84-89). The second method, the 'self penalty walk' algorithm (SPW), refines an initially guessed path by minimizing the sum of the energies of its structures (Elber,R. and Karplus,M., Chem. Phys. Lett. (1987) 139, 375-380). Thus, starting from a linear path as a first approximation, it produces a reaction coordinate of the transition. The structures of the TMD and SPW paths are similar only in the beginning while the middle part of the SPW path links the two TMD branches. The activation of alpha-chymotrypsin in the TMD path starts with a movement of loop VII (residues 215-225), pulling on loop VI (residues 186-194). Then the side chain of Met192 turns to the surface and Ile16 approaches Asp194 to form a salt bridge. In the TMD deactivation path, loop VII also moves and pushes loop VI to the protein core. The Met192 side chain adopts three intermediate conformations, till the salt bridge Ile16-Asp194 is broken and loop VI rearranges to its final conformation. In the SPW pathway both the formation of the salt bridge and the movement of Met192 happen simultaneously between two consecutive steps.

Molecular mechanisms of pressure induced conformational changes in BPTI.

We have performed a 800 ps molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) in water coupled to a pressure bath at 1, 10,000, 15,000, and 20,000 bar. The simulation reproduces quite well the experimental behavior of the protein under high pressure. The protein keeps its globular form, but adopts a different conformation with a very small reduction in volume. Some residues in the hydrophobic core become exposed to water and a large part of the secondary structure of the protein, (60% of the sheet structure and 40% of the helical structure) is denatured between 10 and 15 kbar. This is in good agreement with experimental data (Goossens, K., et al. Eur. J. Biochem, 236:254-262, 1996) that show denaturation of BPTI between 8 and 14 kbar. A further increase of the pressure results in a freezing of the protein as deduced from the large decrease of the mobility of the residues. During the simulation, the normal structure of water changes from an ice Ih-like to an ice VI-like structure, while keeping the liquid state. The driving force of the high pressure induced conformational transition seems be the higher compressibility of the water compared with the protein. This produces a change in the solvent properties and leads to penetration of the solvent into the hydrophobic core.

Molecular dynamics simulation of the solution structures of Ha-ras-p21 GDP and GTP complexes: flexibility, possible hinges, and levers of the conformational transition.

Unconstrained molecular dynamics simulations of the GDP and GTP complexes of Ha-ras p21 protein are performed in aqueous environment for 500 ps, using the GROMOS force field. The solvated structures are mutually compared as well as to the X-ray structures [Tong, L. A., de Vos, A. M., Milburn, M. V., & Kim, S. H. (1991) J. Mol. Biol. 217, 503-516; Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsh, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The simulations show areas of flexibility, with deviations from the original structures. The parts that show differences between the two solvated forms are those from residues 12 to 17, 25 to 38, 41 to 51, 57 to 73, 99 to 112, and 120 to 152, coincident with areas of flexibility. Some of these areas also show differences between the X-ray structures and are part of loops on the surface of the protein. Many of the residues in the ends of these loops undergo dihedral transitions during the solvation process. Of all the dihedral transitions observed, 62% occur around the ends of these loops. This suggests that the ends of the areas from 12 to 17, 25 to 38, and 57 to 73 are the hinge points of the conformational transition between the GTP and the GDP forms. The study of the nucleotide interactions in the solution forms shows that residues 29, 30, and 35 establish contacts with the gamma-phosphate and the sugar ring of the GTP and thus these contacts could be proposed as the possible levers of the conformational transition that accompanies GTP hydrolysis.

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.

Structure-function analysis of human interleukin-6. Evidence for the involvement of the carboxy-terminus in function.

C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% beta-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% beta-structure, and 18% turn and coil.