Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis.

BACKGROUND: We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. METHODOLOGY/RESULTS: Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. CONCLUSION: Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking.

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

Label-free screening of drug-protein interactions by time-resolved Fourier transform infrared spectroscopic assays exemplified by Ras interactions.

Time-resolved Fourier transform infrared (FT-IR) spectroscopy can reveal molecular details of protein interactions. Analysis of difference spectra selects the absorptions of respective protein groups involved in an interaction against the background of the whole sample. By comparison of the same difference spectrum with and without a small molecule, one can determine whether the small molecule interferes with the protein or not. Usually a marker band of a specific residue of the protein is monitored. Here, we show three different time-resolved FT-IR assays detecting interactions of potential small molecules for molecular therapy with the GTPase Ras as an example for small GTPase binding proteins. Ras regulates signal transduction processes through a switching mechanism, cycling between an active "on" GTP-bound form and an inactive "off" GDP-bound state. Molecular defects in Ras can impair the ability of Ras and the Ras-RasGAP complex to hydrolyze GTP, contributing to uncontrolled cell growth and cancer. Oncogenic mutated Ras is found in about 30% of all cancer cells. We show in vitro assays, indicating (I) the shift of Ras into its "off" conformation, which inhibits the Ras pathway; (II) down-regulation of Ras signaling by changes in the Ras-Raf effector interaction; and (III) down-regulation of Ras signaling pathway by catalyzing GTP hydrolysis. Since almost all molecules have characteristic marker bands in the infrared, time-resolved FT-IR spectroscopy can be used label-free. No artificial nucleotides that could influence the interaction are needed. Both, sample preparation and evaluation can be automated in order to allow for high-throughput screening.

Direct evidence for the function of FUM13 in 3-ketoreduction of mycotoxin fumonisins in Fusarium verticillioides.

Fumonisins are mycotoxins produced by Fusarium verticillioides, a widespread pathogen of corn. Although the gene cluster for the biosynthesis of fumonisins has been cloned, the majority of the genes have not been biochemically characterized. Here, we report the biochemical characterization of FUM13, a gene that encodes a short-chain dehydrogenase/reductase required for fumonisin biosynthesis. FUM13 has been expressed in E. coli, and the produced protein, Fum13p, has been purified. When the protein was incubated with 3-keto fumonisin B(3) (FB(3)) in the presence of NADPH, FB(3) was produced. The data provide direct evidence for the role of FUM13 in the 3-ketoreduction of fumonisins. In a functional complementation experiment, FUM13 gene was introduced into tsc10 mutants of the yeast Saccharomyces cerevisiae, which carry a mutation in the 3-ketosphinganine reductase gene in the sphingolipid pathway. The tsc10 mutants were not able to grow on the selection medium, but the same mutants transformed with FUM13 were able to grow. The results further confirm the function of FUM13 in 3-ketoreduction in vivo.

Determining the biosynthetic sequence in the early steps of the fumonisin pathway by use of three gene-disruption mutants of Fusarium verticillioides.

Fumonisins are polyketide-derived mycotoxins produced by Fusarium verticillioides, a fungal pathogen of corn plants. Although a gene cluster for the biosynthesis of fumonisins has been cloned, the biosynthetic pathway is still not clear. We have used three gene-disrupted mutants, designated DeltaFUM1, DeltaFUM6, and DeltaFUM8, to study the early steps of the pathway. Fumonisins were not produced in single-strain cultures of the DeltaFUM1, DeltaFUM6, and DeltaFUM8 mutants. However, fumonisins were produced by DeltaFUM1 or DeltaFUM8 mutants when they were cocultured with the DeltaFUM6 mutant. No fumonisins were produced when the DeltaFUM1 and DeltaFUM8 mutants were cocultured. These results suggest that the DeltaFUM6 mutant produces a fumonisin intermediate that can be further metabolized by fumonisin biosynthetic enzymes in the DeltaFUM1 and DeltaFUM8 mutants. To isolate the potential intermediates produced by DeltaFUM6, we followed a time course of cocultures of the DeltaFUM1 and DeltaFUM6 and the DeltaFUM8 and DeltaFUM6 mutants. Liquid chromatographic-mass spectrometric data suggested that metabolites having the general carbon skeleton of fumonisins with 1-4 hydroxyl groups were accumulated over a 7-day period. These results indicate that fumonisin biosynthesis starts with Fum1p-catalyzed carbon-chain assembly followed by the Fum8p-catalyzed alanine condensation. The resulting product then can be further oxidized by Fum6p and other enzymes.

Fum3p, a 2-ketoglutarate-dependent dioxygenase required for C-5 hydroxylation of fumonisins in Fusarium verticillioides.

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species. The B series fumonisins, FB(1), FB(2), FB(3), and FB(4), are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae. Yeast cells expressing the Fum3p converted FB(3) to FB(1), indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe(2+), ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.