Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF.

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation.

B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes.

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.

The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone and its implications for the activation mechanism.

The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33.

Cleavage of bacteriophage lambda cI repressor involves the RecA C-terminal domain.

The SOS response to DNA damage in Escherichia coli involves at least 43 genes, all under the control of the LexA repressor. Activation of these genes occurs when the LexA repressor cleaves itself, a reaction catalyzed by an active, extended RecA filament formed on DNA. It has been shown that the LexA repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. Bacteriophages, such as lambda, have repressors (cI) that are structural homologs of LexA and also undergo self-cleavage when SOS is induced. It has been puzzling that some mutations in RecA that affect the cleavage of repressors are in the C-terminal domain (CTD) far from the groove where cleavage is thought to occur. In addition, it has been shown that the rate of cleavage of cI by RecA is dependent upon both the substrate on which RecA is polymerized and the ATP analog used. Electron microscopy and three-dimensional reconstructions show that the conformation and dynamics of RecA's CTD are also modulated by the polynucleotide substrate and ATP analog. Under conditions where the repressor cleavage rates are the highest, cI is coordinated within the groove by contacts with RecA's CTD. These observations provide a framework for understanding previous genetic and biochemical observations.

The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA.

The DH-PH domain tandems of Dbl-homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho-family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH-PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH-PH tandem of RhoA-specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH-PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide-free RhoA exhibits elevated dynamics when in complex with DH-PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.

Divergence of quaternary structures among bacterial flagellar filaments.

It has been widely assumed that the atomic structure of the flagellar filament from Salmonella typhimurium serves as a model for all bacterial flagellar filaments given the sequence conservation in the coiled-coil regions responsible for polymerization. On the basis of electron microscopic images, we show that the flagellar filaments from Campylobacter jejuni have seven protofilaments rather than the 11 in S. typhimurium. The vertebrate Toll-like receptor 5 (TLR5) recognizes a region of bacterial flagellin that is involved in subunit-subunit assembly in Salmonella and many other pathogenic bacteria, and this short region has diverged in Campylobacter and related bacteria, such as Helicobacter pylori, which are not recognized by TLR5. The driving force in the change of quaternary structure between Salmonella and Campylobacter may have been the evasion of TLR5.

Structure of the Bacillus subtilis OhrB hydroperoxide-resistance protein in a fully oxidized state.

The crystal structure of the fully oxidized form of the Bacillus subtilis organic hydroperoxide-resistance (OhrB) protein is reported at 2.1 A resolution. The electron density reveals an intact catalytic disulfide bond (Cys55-Cys119) in each of the two molecules, which are intertwined into a canonical obligate dimer. However, the stereochemistry of the disulfides is unorthodox and strained, suggesting that they are sensitive to reducing agents. A deep solvent-accessible gorge reaching Cys55 may represent the access route for the reductant.