Nearest-cell: a fast and easy tool for locating crystal matches in the PDB.

When embarking upon X-ray diffraction data collection from a potentially novel macromolecular crystal form, it can be useful to ascertain whether the measured data reflect a crystal form that is already recorded in the Protein Data Bank and, if so, whether it is part of a large family of related structures. Providing such information to crystallographers conveniently and quickly, as soon as the first images have been recorded and the unit cell characterized at an X-ray beamline, has the potential to save time and effort as well as pointing to possible search models for molecular replacement. Given an input unit cell, and optionally a space group, Nearest-cell rapidly scans the Protein Data Bank and retrieves near-matches.

SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new technologies and future aspects.

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future.

SPINE workshop on automated X-ray analysis: a progress report.

The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Gene polymorphism in Netherton and common atopic disease.

Atopic dermatitis (AD) and asthma are characterized by IgE-mediated atopic (allergic) responses to common proteins (allergens), many of which are proteinases. Loci influencing atopy have been localized to a number of chromosomal regions, including the chromosome 5q31 cytokine cluster. Netherton disease is a rare recessive skin disorder in which atopy is a universal accompaniment. The gene underlying Netherton disease (SPINK5) encodes a 15-domain serine proteinase inhibitor (LEKTI) which is expressed in epithelial and mucosal surfaces and in the thymus. We have identified six coding polymorphisms in SPINK5 (Table 1) and found that a Glu420-->Lys variant shows significant association with atopy and AD in two independent panels of families. Our results implicate a previously unrecognized pathway for the development of common allergic illnesses.

Mutations at amino acid positions 63, 189, and 396 of human immunodeficiency virus type 1 reverse transcriptase (RT) partially restore the DNA polymerase activity of a Trp229Tyr mutant RT.

Trp229 is part of the nonnucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV-1 reverse transcriptase (RT). It is also an important constituent of the so-called "primer grip." Using a recombinant virus assay, we tried to obtain recombinant virus containing a Trp229Phe or a Trp229Tyr mutation in its RT. Previous studies already established the very low DNA polymerase activities of both the Trp229Phe and the Trp229Tyr mutant RT enzymes. We were able to obtain a Trp229Tyr but not a Trp229Phe mutant virus. However, in addition to the Trp229Tyr mutation this mutant virus also contained an Ile63Met, a Val189Ile, and a Glu396Gly mutation in its RT. When we evaluated the quadruple mutant virus for sensitivity/resistance against a variety of NNRTIs, no significant difference with the sensitivity/resistance profile of the single Trp229Tyr mutant RT enzyme could be observed. We found that the three additional mutations partly restored the low RNA- and DNA-dependent DNA polymerase activities of the Trp229Tyr mutant enzyme. Kinetic analysis revealed that both template/primer binding and dNTP incorporation are affected by the Trp229Tyr mutation. Our findings demonstrate that a mutation at position 229 is unlikely to occur under NNRTI drug pressure due to the poor catalytic activity of the singly mutated RT and the favorable drug sensitivity profile of the mutated enzyme/viruses in both the absence and the presence of the compensatory mutations. Therefore, amino acid position 229 may be regarded as an excellent amino acid target within the NNRTI pocket for rational drug design.

Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype.

OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.

Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase.

The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-beta-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil].

Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of alanine-167 to tyrosine.

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced approximately 2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.

Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.

Solution structure of a HNA-RNA hybrid.

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

Presence of 2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxath iole-2",2"-dioxide) (TSAO)-resistant virus strains in TSAO-inexperienced HIV patients.

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Mutational analysis of trp-229 of human immunodeficiency virus type 1 reverse transcriptase (RT) identifies this amino acid residue as a prime target for the rational design of new non-nucleoside RT inhibitors.

Trp-229 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV type 1 (HIV-1) reverse transcriptase (RT), and is also part of the "primer grip" of HIV-1 RT. Using site-directed mutagenesis, seven RT mutants were constructed bearing the mutations 229Phe, 229Tyr, 229Ile, 229His, 229Lys, 229Cys, and 229Gln. We found that all of the mutants showed severely compromised RNA- and DNA-dependent DNA polymerase activities (<2% of wild-type activity). The recombinant 229Phe and 229Tyr RT enzymes were among the mutant enzymes with the highest activity (0.7 and 1.1% of wild-type activity, respectively) and we evaluated these for resistance against several NNRTIs. No resistance was found for the 229Phe RT, but the 229Tyr RT showed a approximately 20-fold resistance against UC-781 and lower resistance against emivirine and nevirapine. Attempts to make recombinant virus strains bearing the single 229Phe or 229Tyr RT mutation failed. Experiments in which we varied the pentenyl ether substituent of the thiocarboxanilide UC-781 revealed that Trp-229 can be specifically targeted by NNRTIs and that an alkenyloxy group length of five atoms assures an optimal interaction of the thiocarboxanilides with Trp-229. Our findings indicate that Trp-229, when combined with other crucial immutable amino acids (i.e., Tyr-318), is an appropriate candidate for the targeted design of new NNRTIs.

Design, synthesis, and enzymatic evaluation of multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase.

A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase. The compounds synthesized include 1-(phosphonoalkyl)thymines with six to nine methylenes (1-4, respectively); 1-[(Z)-4-phosphonomethoxy-2-butenyl]thymine (5) and its butyl and 2,3-cis-dihydroxybutyl derivatives (6 and 7, respectively); 1-[(Z)-(4-(phosphonomethoxy)methoxy)-2-butenyl]thymine (8) and also its butyl and 2,3-cis-dihydroxybutyl analogues (9 and 10); and 1-[((Z)-4-(phosphonomethoxy)-2-butenoxy)methyl]thymine (11). Evaluation of these compounds against E. coli revealed significant enzymatic inhibition by 2, 3, 4, 6, and 8 at a concentration of 1000 microM, 3 and 4 being the most potent. Replacement of the thymine base in 3 by 6-amino-5-bromouracil and 7-deazaxanthine afforded compounds 12 and 13, which showed a pronounced improvement of TPase inhibition, comparable to 7-deazaxanthine. When inorganic phosphate was used as a variable substrate, compounds 12 and 13 displayed competitive kinetics with respect to phosphate, indicating a direct interaction of these compounds with the phosphate binding site. Also compounds 12 and 13 were found to be competitive inhibitors of TPase against thymidine as a variable substrate. These results are consistent with the compounds being multisubstrate analogue inhibitors of E. coli TPase, and they represent the first example of such TPase inhibitors.

Resistance of herpes simplex virus type 1 against different phosphonylmethoxyalkyl derivatives of purines and pyrimidines due to specific mutations in the viral DNA polymerase gene.

Drug-resistant strains of herpes simplex virus type 1 (HSV-1) were selected under the pressure of (S)-3-hydroxy-2-phosphonylmethoxypropyl (HPMP) derivatives of cytosine (HPMPC, cidofovir) and adenine (HPMPA) and 2-phosphonylmethoxyethyl (PME) derivatives of adenine (PMEA, adefovir) and 2,6-diaminopurine (PMEDAP). HPMPC-resistant (HPMPC(r)) and HPMPA(r) strains were cross-resistant to one another, but they remained sensitive to foscarnet (PFA), acyclovir (ACV) and the PME derivatives, while the PMEA(r) and PMEDAP(r) strains showed cross-resistance to PFA and ACV. The PMEA(r), PMEDAP(r) and PFA(r) mutants all revealed a single nucleotide change resulting in a Ser-724 to Asn mutation within the conserved region II of the DNA polymerase. Two HPMPA(r) clones and one HPMPC(r) clone possessed single amino acid changes in the DNA polymerase (HPMPA(r) clone D1, Leu-1007 to Met; HPMPA(r) clone B5, Ile-1028 to Thr; HPMPC(r) clone C3, Val-573 to Met). The HPMPC(r) clone A4 contained two mutations, Ala-136 to Thr and Arg-700 to Met. The mutation at position 136, located outside the catalytic domain of the enzyme, was not detected in other HPMPC(r) clones, suggesting that this mutation may not be responsible for the resistant phenotype. Residue 573 is located within the 3'-->5' exonuclease editing domain close to the catalytically important residues Tyr-577 and Asp-581. Similarly, residue 700 is located in the palm subdomain of the catalytic domain, adjacent to the Asp residues 717, 886 and 888 that are vital for polymerase activity. The HPMPA(r) mutations at residues 1007 and 1028, beyond the last conserved region, still fall within the thumb subdomain of the catalytic domain. The different drug-resistant mutants varied in neurovirulent behaviour, the HPMPC(r) strains showing reduced neurovirulence compared with the wild-type.

Phenylethylthiazolylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and HIV-2 reverse transcriptases. Structural and biochemical analyses.

Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC(50) of 6 nM but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC(50) of 5 nM) and also inhibits HIV-2 RT (IC(50) of 2.2 microM). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC).(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1.

Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies.

We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.

Characterization of multiple nuclear localization signals in herpes simplex virus type 1 thymidine kinase.

We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.

Modulation of fibroblast growth factor-2 receptor binding, signaling, and mitogenic activity by heparin-mimicking polysulfonated compounds.

Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.

Further additions to MolScript version 1.4, including reading and contouring of electron-density maps.

MolScript is one of the most popular programs for the generation of publication-quality figures and the recent re-working of the program should ensure its continued popularity. However, some functionality of particular interest to crystallographers is not part of the standard program. A modified MolScript version 1.4 has been described previously, with more flexible colouring schemes among its new features. This report describes further enhancements to MolScript version 1.4, including facilities for drawing rods for helices and ribbons for oligonucleotides and allowing several formats of electron-density maps to be read and contoured using either lines or smoothed triangulated surfaces.