Expression, purification, and crystallization of the RGS-like domain from the Rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach

Lsc-homology domains are found in several eukaryotic nucleotide exchange factors which act on Rho-family GTPases. They show limited amino acid sequence similarity to RGS proteins, which down-regulate the cellular signaling by the alpha-subunits of trimeric G-proteins and have been shown to interact with Galpha12 and Galpha13. It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases. We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues. Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source. The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit.     

The histone H1 C-terminal domain binds to the apoptotic nuclease, DNA fragmentation factor (DFF40/CAD) and stimulates DNA cleavage

The apoptotic nuclease, DNA fragmentation factor (DFF40/CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of programmed cell death. Previously, we demonstrated that histone H1 greatly stimulates naked DNA cleavage by this nuclease. Here, we investigate the mechanism of this stimulation with native and recombinant mouse and human histone H1 species. Using a series of truncation mutants of recombinant histone H1-0, we demonstrate that the H1 C-terminal domain (CTD) is responsible for activation of DFF40/CAD. We show further that the intact histone H1-0 CTD and certain synthetic CTD fragments bind to DFF40/CAD and confer upon it an increased ability to bind to DNA. Interestingly, we find that each of the six somatic cell histone H1 isoforms, whose CTDs differ significantly in primary sequence but not amino acid composition, equally activate DFF40/CAD. We conclude that the interactions identified here between the histone H1 CTD and DFF40/CAD target and activate linker DNA cleavage during the terminal stages of apoptosis.     

Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg²⁺, exhibits a trace of activity in the presence of Mn²⁺, is not co-stimulated by Ca²⁺, is inhibited by Zn²⁺ or Cu²⁺, and has high activity over a rather broad pH range (7.0–8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K⁺] of 50–125 mM, which is in the range of the cytoplasmic [K⁺] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K⁺]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA.     

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.     

Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis

DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.     

Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells.     

Acid-base catalysis in the argininosuccinate lyase reaction

The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.     

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.     

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.