The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

More hydrogen bonds for the (structural) biologist.

Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.

The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Regulatory GTPases.

The past year has witnessed a tremendous increase in our understanding of the structures and interactions of the GTPases. The highlights include crystal structures of G alpha subunits, as well as the first complex between a GTPase (Rap1A) and an effector molecule (c-Raf1 Ras-binding domain). In the field of elongation factors (EFs), three very important structures have been determined: EF-G, the ternary complex of EF-Tu.GTP with aminoacyl-tRNA, and the EF-Tu.EF-Ts complex.

An alpha to beta conformational switch in EF-Tu.

BACKGROUND: The bacterial elongation factor EF-Tu recognizes and transports aminoacyl-tRNAs to mRNA-programmed ribosomes. EF-Tu shares many structural and functional properties with other GTPases whose conformations are regulated by guanine nucleotides. RESULTS: An intact form of Escherichia coli EF-Tu complexed with GDP has been crystallized in the presence of the EF-Tu-specific antibiotic GE2270 A. The three-dimensional structure has been solved by X-ray diffraction analysis and refined to a final crystallographic R factor of 17.2% at a resolution of 2.5 A. The location of the GE2270 A antibiotic-binding site could not be identified. CONCLUSIONS: The structure of EF-Tu-GDP is nearly identical to that of a trypsin-modified form of EF-Tu-GDP, demonstrating conclusively that the protease treatment had not altered any essential structural features. The present structure represents the first view of an ordered Switch I region in EF-Tu-GDP and reveals similarities with two other GTPases complexed with GDP: Ran and ADP-ribosylation factor-1. A comparison of the Switch I regions of the GTP and GDP forms of EF-Tu also reveals that a segment, six amino acids in length, completely converts from an alpha helix in the GTP complex to beta secondary structure in the GDP form. The alpha to beta switch in EF-Tu may represent a prototypical activation mechanism for other protein families.

Non-proline cis peptide bonds in proteins.

In a non-redundant set of 571 proteins from the Brookhaven Protein Data Base, a total of 43 non-proline cis peptide bonds were identified. Average geometrical parameters of the well-defined cis peptide bonds in proteins determined at high resolution show that some parameters, most notably the bond angle at the amide bond nitrogen, deviate significantly from the corresponding one in the trans conformation. Since the same feature was observed in cis amide bonds in small molecule structures found in the Cambridge Structural Data Base, a new set of parameters for the refinement of protein structures containing non-Pro cis peptide bonds is proposed.A striking preference was observed for main-chain dihedral angles of the residues involved in cis peptide bonds. All residues N-terminal and most residues C-terminal to a non-Pro cis peptide bond (except Gly) are located in the beta-region of a phi/psi plot. Also, all of the few C-terminal residues (except Gly) located in the alpha-region of the phi/psi plot constitute the start of an alpha-helix in the respective structure. In the majority of cases, an intimate side-chain/side-chain interaction was observed between the flanking residues, often involving aromatic side-chains. Interestingly, most of the cases found occur in functionally important regions such as close to the active site of proteins. It is intriguing that many of the proteins containing non-proline cis peptide bonds are carbohydrate-binding or processing proteins. The occurrence of these unusual peptide bonds is significantly more frequent in structures determined at high resolution than in structures determined at medium and low resolution, suggesting that these bonds may be more abundant than previously thought. On the basis of our experience with the structure determination of coagulation factor XIII, we developed an algorithm for the identification of possibly overlooked cis peptide bonds that exploits the deviations of geometrical parameters from ideality. A few likely candidates based on our algorithm have been identified and are discussed.

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3.2 A resolution.

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3.2 A resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0.857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of alpha-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin. Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.

C-H...pi-interactions in proteins.

A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.

Crystals of intact elongation factor Tu from Thermus thermophilus diffracting to high resolution.

The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution. They exhibit space group C2, with a = 150.3(6) A, b = 99.6(3) A, c = 40.1(1) A, beta = 95.4(2) degrees, and contain one elongation factor Tu molecule per asymmetric unit.

Structure and expression of elongation factor Tu from Bacillus stearothermophilus.

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.

GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes.

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Adhesion receptors in malignant transformation and dissemination of gastrointestinal tumors.

Changes in the expression and function of adhesion molecules on the surface of cancer cells are important characteristics in the development of gastrointestinal malignancies and might be used in the future as prognostic factors or as new targets for diagnostic and therapeutic approaches. In esophageal cancer a down-regulation of the E-cadherin receptor and the cytoplasmic protein alpha-catenin is associated with tumor dedifferentiation, infiltrative growth and lymph-node metastasis. In gastric cancer a reduction of E-cadherin expression due to gene mutations is restricted to diffuse-type tumors while the occurrence of the CD44-standard and the CD44-9v isoform is significantly related to a higher tumor-induced mortality and a shorter survival time. The CD44-6v isoform is predominantly expressed by intestinal-type gastric carcinomas, giving these tumor cells the ability to perform lymph-node metastasis. In pancreatic cancer the expression of integrin adhesion receptors is significantly altered during the malignant transformation while a loss of the E-cadherin receptor can generate dedifferentiation and invasiveness of pancreas carcinoma cells. There is increasing evidence that integrin receptors as well as different isoforms of the CD44 receptor are altered following the malignant transformation of colonic mucosa into adenomas and invasive carcinomas. The expression of the CD44-6v isoform seems to be associated with an adverse prognosis in colorectal cancer due to the development of tumor metastases. A strong correlation has been observed between the expression of the 67-kDa laminin receptor and the degree of differentiation, the invasive phenotype and the metastatic abilities af colorectal cancer cells. Analyzing the expression of the E-cadherin receptor showed that this receptor may serve as an independent prognostic marker in Dukes' stage B colorectal cancer to identify patients with poor prognosis and designate them for intensive adjuvant therapy and clinical observation after curative surgical tumor treatment.

Inhibitory mechanisms of antibiotics targeting elongation factor Tu.

Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal structure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided structural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy.

The selectivity filter of a ligand-gated ion channel. The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor.

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.

Two non-proline cis peptide bonds may be important for factor XIII function.

The structure of recombinant human cellular factor XIII zymogen was solved in its monoclinic crystal form and refined to an R-factor of 18.3% (Rfree = 23.6%) for all data between 40.0 and 2.1 A resolution. Two non-proline cis peptide bonds were detected. One is between Arg310 and Tyr311 close to the active site cysteine residue (Cys314) and the other is between Gln425 and Phe426 at the dimerization interface. The structure and the role of these cis peptides are discussed in the light of their possible importance for factor XIII function.

A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria.

The crystal structure of the DNA-binding domain of E. coli SSB (EcoSSB) has been determined to a resolution of 2.5 A. This is the first reported structure of a prokaryotic SSB. The structure of the DNA-binding domain of the E. coli protein is compared to that of the human mitochondrial SSB (HsmtSSB). In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity. EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer. This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer. Crucial differences in the dimer-dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross-species heterotetramers, in contrast to many bacterial SSBs.

Crystallization of blood coagulation factor XIII by an automated procedure.

Both recombinant blood coagulation factor XIII alpha-chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X-ray analysis.

Phase II trial of N-(phosphonacetyl)-L-aspartate (PALA), 5-fluorouracil and recombinant interferon-alpha-2b in patients with advanced gastric carcinoma.

The aspartate transcarbamoylase inhibitor, N-(phosphonacetyl)-L-aspartate (PALA), synergistically enhanced the cytotoxicity of a combination of 5-fluorouracil (5-FU) and interferon-alpha (IFN) against human colon cancer cell lines in vitro. To test the efficacy of this combination in the clinical setting, patients with locally advanced or advanced gastric carcinoma were treated with the combination of PALA, 5-FU and IFN (PFI). Patients were required to have biopsy-proven disease beyond the scope of surgical resection, measurable disease, no prior chemotherapy, adequate bone marrow, renal and hepatic function, to be fully ambulatory and to have given informed consent. Drug was administered as follows: PALA, 250 mg/m2, 15 min i.v. infusion, days 1, 15, 22, 29, and then weekly; 5-FU, 750 mg/m2 daily x 5 as a continuous i.v. infusion beginning day 2, then at 750 mg/m2 days 16, 23 and 30, then weekly; IFN, 9 MU subcutaneously three times per week beginning day 2. There were 22 patients enrolled. The major toxicities were fatigue and associated neurotoxicity, with acceptable gastrointestinal and haematological toxicities. There was one complete responder (5%) and 3 partial responders (14%); two of these responses were durable (> 3 years). Despite this modest clinical activity, other regimens for advanced gastric cancer such as FAMTX and ELF appear to have greater activity with comparable toxicity.

Structural model for the selenocysteine-specific elongation factor SelB.

A structural model was established for the N-terminal part of translation factor SelB which shares sequence similarity with EF-Tu, taking into account the coordinates of the EF-Tu 3D structure and the consensus of SelB sequences from four bacteria. The model showed that SelB is homologous in its N-terminal domains over all three domains of EF-Tu. The guanine nucleotide binding site and the residues involved in GTP hydrolysis are similar to those of EF-Tu, but with some subtle differences possibly responsible for the higher affinity of SelB for GTP compared to GDP. In accordance, the EF-Tu epitopes interacting with EF-Ts are lacking in SelB. Information on the formation of the selenocysteyl-binding pocket is presented. A phylogenetic comparison of the SelB domains homologous to EF-Tu with those from EF-Tu and initiation factor 2 indicated that SelB forms a separate class of translation factors.

Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe).

The effect of aminoacylation and ternary complex formation with elongation factor Tu.GTP on the tertiary structure of yeast tRNA(Phe) was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNA(Phe) does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA(Phe) with elongation factor Tu.GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA(Phe) upon binding to elongation factor Tu.GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA(Phe)-elongation factor Tu-GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA(Phe) preserves its typical L-shaped tertiary structure in the complex. At high (> 10(-4) M) ligand concentrations a complex between tRNA(Phe) and elongation factor Tu-GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA(Phe) and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.