Structure-function characterization of an insecticidal protein GNIP1Aa, a member of an MACPF and ß-tripod families.

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here ß-tripod. Using mutagenesis, we identified functionally important regions in the ß-tripod domain, which may be involved in target recognition.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments.

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.

FtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division.

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.

Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2.

FtsZ from most bacteria assembles rapidly in vitro, reaching a steady-state plateau in 5-10 s after addition of GTP. A recent study used a novel dynamic light-scattering technique to assay the assembly of FtsZ from Caulobacter crescentus (CcFtsZ) and reported that assembly required 10 min, ∼100 times slower than for related bacteria. Previous studies had indicated normal, rapid assembly of CcFtsZ. We have reinvestigated the assembly kinetics using a mutant L72W, where assembly of subunits into protofilaments results in a significant increase in tryptophan fluorescence. We found that assembly reached a plateau in 5-10 s and showed no change in the following 10 min. This was confirmed by 90° light scattering and negative-stain electron microscopy. The very slow kinetics in the dynamic light-scattering study may be related to a refractory state induced when the FtsZ protein is stored without nucleotide, a phenomenon that we had observed in a previous study of EcFtsZ. We conclude that CcFtsZ is not an outlier, but shows rapid assembly kinetics similar to FtsZ from related bacteria.

SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid.

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.

Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.

FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.

SulA inhibits assembly of FtsZ by a simple sequestration mechanism.

We have investigated the inhibition by SulA of the assembly of Escherichia coli FtsZ. Using quantitative GTPase and fluorescence assays, we found that SulA inhibition resulted in an increase in the apparent critical concentration for FtsZ assembly. The increase in apparent critical concentration was always less than the total amount of SulA added, suggesting that the association of SulA and FtsZ was of modest affinity. Isothermal titration calorimetry gave a value of 0.78 µM for the dissociation constant of the FtsZ-SulA complex, similar in magnitude to the 0.72 µM critical concentration of FtsZ protofilament assembly at steady state. We modeled the reaction as an equilibrium competition between (a) FtsZ subunits assembling onto protofilaments or (b) binding SulA. When FtsZ was assembled in GMPCPP or in EDTA, the inhibition by SulA was reduced. The reduced inhibition could be explained by a 3- and 10-fold weaker binding of SulA to FtsZ. The mutant D212G, which has no GTPase activity and therefore minimal subunit cycling, was shown here to assemble one-stranded protofilaments, and the assembly was blocked by SulA. We also assayed the SulA and FtsZ proteins from Pseudomonas. The SulA inhibition was stronger than with the E. coli proteins, and the model indicated a 5-fold higher affinity of Pseudomonas SulA for FtsZ.

Folding and assembly kinetics of procaspase-3.

Caspases are vital to apoptosis and exist in the cell as inactive zymogens. Dimerization is central to procaspase activation because the active sites are comprised of loops from both monomers. Although initiator procaspases are stable monomers until activated on cell death scaffolds, the effector caspases, such as procaspase-3, are stable dimers. The activation mechanisms are reasonably well understood in terms of polypeptide chain cleavage and subsequent active site rearrangements in the dimer, but the mechanisms that govern dimer assembly are not known. To further understand procaspase dimerization, we examined the folding and assembly of procaspase-3 by fluorescence emission, circular dichroism, differential quenching by acrylamide, anisotropy, and enzyme activity assays. Single-mixing stopped-flow refolding studies showed a complex burst phase in which multiple monomeric species form rapidly. At longer times, the monomer folds through several intermediates, some of which appear to be off-pathway or misfolded, before eventually forming a dimerization-competent species. Enzyme activity studies demonstrated a slow rate of dimerization (approximately 70 M(-1) s(-1)). In addition, single-mixing stopped-flow unfolding studies revealed a complex unfolding process with a slow rate of dimer dissociation. Interestingly, multiple dimeric species were observed in the burst phase for unfolding, suggesting that the native ensemble consists of at least two major conformations. Collectively, these results demonstrate complex folding and unfolding behavior for procaspase-3 and suggest that slow dimerization results from the lack of stabilizing native contacts in the initial encounter complex.

Practical approaches to protein folding and assembly: spectroscopic strategies in thermodynamics and kinetics.

We describe here the use of several spectroscopies, such as fluorescence emission, circular dichroism, and differential quenching by acrylamide, in examining the equilibrium and kinetic folding of proteins. The first section regarding equilibrium techniques provides practical information for determining the conformational stability of a protein. In addition, several equilibrium-folding models are discussed, from two-state monomer to four-state homodimer, providing a comprehensive protocol for interpretation of folding curves. The second section focuses on the experimental design and interpretation of kinetic data, such as burst-phase analysis and exponential fits, used in elucidating kinetic folding pathways. In addition, simulation programs are used routinely to support folding models generated by kinetic experiments, and the fundamentals of simulations are covered.

Rapid folding and unfolding of Apaf-1 CARD.

Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an alpha-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.