Structures of inactive CRP species reveal the atomic details of the allosteric transition that discriminates cyclic nucleotide second messengers.

The prokaryotic global transcription factor CRP has been considered to be an ideal model for in-depth study of both the allostery of the protein and the differential utilization of the homologous cyclic nucleotide second messengers cAMP and cGMP. Here, atomic details from the crystal structures of two inactive CRP species, an apo form and a cGMP-bound form, in comparison with a known active conformation, the cAMP-CRP complex, provide macroscopic and microscopic insights into CRP allostery, which is coupled to specific discrimination between the two effectors. The cAMP-induced conformational transition, including dynamic fluctuations, can be driven by the fundamental folding forces that cause water-soluble globular proteins to construct an optimized hydrophobic core, including secondary-structure formation. The observed conformational asymmetries underlie a negative cooperativity in the sequential binding of cyclic nucleotides and a stepwise manner of binding with discrimination between the effector molecules. Additionally, the finding that cGMP, which is specifically recognized in a syn conformation, induces an inhibitory conformational change, rather than a null effect, on CRP supports the intriguing possibility that cGMP signalling could be widely utilized in prokaryotes, including in aggressive inhibition of CRP-like proteins.

Structural characterization of de novo designed L5K5W model peptide isomers with potent antimicrobial and varied hemolytic activities.

In an effort to develop short antimicrobial peptides with simple amino acid compositions, we generated a series of undecapeptide isomers having the L(5)K(5)W formula. Amino acid sequences were designed to be perfectly amphipathic when folded into a helical conformation by converging leucines onto one side and lysines onto the other side of the helical axis. The single tryptophans, whose positions were varied in the primary structures, were located commonly at the critical amphipathic interface in the helical wheel projection. Helical conformations and the tryptophanyl environments of the 11 L(5)K(5)W peptides were confirmed and characterized by circular dichroism, fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers exhibited a potent, broad-spectrum of antibacterial activity with just a slight variance in individual potency, whereas their hemolytic activities against human erythrocytes were significantly diversified. Interestingly, helical dispositions and fluorescence blue shifts of the peptides in aqueous trifluoroethanol solutions, rather than in detergent micelles, showed a marked linear correlation with their hemolytic potency. These results demonstrate that our de novo design strategy for amphipathic helical model peptides is effective for developing novel antimicrobial peptides and their hemolytic activities can be estimated in correlation with structural parameters.

Crystallization and X-ray data collection of HP0902 from Helicobacter pylori 26695.

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His(6) tag ((H6)HP0902) and the other with a C-terminal His(6) tag (HP0902(H6)). The (H6)HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902(H6) crystal belonged to space group P4(3)2(1)2 or P4(1)2(1)2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.

Structural overview on the allosteric activation of cyclic AMP receptor protein.

Cyclic AMP receptor protein (CRP) is a prokaryotic global transcription regulator that controls the expression of nearly 200 genes. The protein, allosterically activated by cAMP binding, binds to DNA and interacts with RNA polymerase. Current understanding on the allosteric process of the Escherichia coli CRP activation can be summarized into a rigid-body movement that involves subunit realignment and domain rearrangement. The main consequence of that overall transition is protrusion and adjustment of F-helices that recognize specific DNA sites. Although physicochemical and structural studies during the past decades have contributed to a comprehensive understanding of the CRP allostery, a paucity of structural information about the cAMP-free form (apo-CRP) has precluded a definite elucidation of the allosterism. In this respect, recent achievements of structures on other CRP-family proteins provide useful information to fill in the details of the allosteric transition of CRP. Thus, in this paper, accomplishments of CRP-family structures are summarized and inspected comparatively with new findings. This review not only provides a structural overview on the allosteric conformational change of CRP but also suggests a thoughtful discussion about unsolved issues or conflicting arguments. Solving those issues and the apo-CRP structure would enable us to finally define the CRP allostery.

Unique Unfoldase/Aggregase Activity of a Molecular Chaperone Hsp33 in its Holding-Inactive State.

The various chaperone activities of heat shock proteins contribute to ensuring cellular proteostasis. Here, we demonstrate the non-canonical unfoldase activity as an inherent functionality of the prokaryotic molecular chaperone, Hsp33. Hsp33 was originally identified as a holding chaperone that is post-translationally activated by oxidation. However, in this study, we verified that the holding-inactive reduced form of Hsp33 (RHsp33) strongly bound to the translational elongation factor, EF-Tu. This interaction was critically mediated by the redox-switch domain of RHsp33 and the guanine nucleotide-binding domain of EF-Tu. The bound RHsp33, without undergoing any conformational change, catalyzed the EF-Tu aggregation by evoking the aberrant folding of EF-Tu to expose hydrophobic surfaces. Consequently, the oligomers/aggregates of EF-Tu, but not its functional monomeric form, were highly susceptible to proteolytic degradation by Lon protease. These findings present a unique example of an ATP-independent molecular chaperone with distinctive dual functions-as an unfoldase/aggregase and as a holding chaperone-depending on the redox status. It is also suggested that the unusual unfoldase/aggregase activity of RHsp33 can contribute to cellular proteostasis by dysregulating EF-Tu under heat-stressed conditions.

Crystal structure and functional characterization of SF216 from Shigella flexneri.

Shigella flexneri is a Gram-negative anaerobic bacterium that causes highly infectious bacterial dysentery in humans. Here, we solved the crystal structure of SF216, a hypothetical protein from the S. flexneri 5a strain M90T, at 1.7 Å resolution. The crystal structure of SF216 represents a homotrimer stabilized by intersubunit interactions and ion-mediated electrostatic interactions. Each subunit consists of three ß-strands and five α-helices with the ß-ß-ß-α-α-α-α-α topology. Based on the structural information, we also demonstrate that SF216 shows weak ribonuclease activity by a fluorescence quenching assay. Furthermore, we identify potential druggable pockets (putative hot spots) on the surface of the SF216 structure by computational mapping.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of SF173 from Shigella flexneri.

Shigella flexneri is a Gram-negative, anaerobic bacterium in the genus Shigella that can cause diarrhoea in humans. SF173, a hypothetical protein from S. flexneri 5a strain M90T, has been cloned, overexpressed, purified and crystallized as a part of laboratory-scale structural genomics project. The SF173 protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.8 M succinic acid pH 7.0 at 293 K. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 1.47 Šresolution and belonged to space group I432, with unit-cell parameters a=b=c=110.245 Å.

Semi-Empirical Structure Determination of Escherichia coli Hsp33 and Identification of Dynamic Regulatory Elements for the Activation Process.

The activation process of the redox-regulated chaperone heat shock protein 33 (Hsp33) is constituted by the oxidation-induced unfolding of the C-terminal zinc-binding domain and concomitant oligomerization of the N-terminal core domain. Herein, the semi-empirical solution structure of Escherichia coli Hsp33 in the reduced, inactive form was generated through conformational space annealing calculations, utilizing minimalistic NMR data and multiple homology restraints. The various conformations of oxidized Hsp33 and some mutant forms were also investigated in solution. Interestingly, a specific region concentrated around the interdomain linker stretch and its interacting counterparts, the N-terminal ß-strand 1 and α-helix 1, hardly showed up as signals in the NMR measurements. The NMR spectra of an Hsp33 derivative with a six-residue deletion in the disordered N-terminus implied a plausible conformational exchange associated with the identified region, and the corresponding exchange rate appeared slower than that of the wild type. Subsequent mutations that destroyed the structure of the ß1 or α1 elements resulted in the formation of a reduced but active monomer, without the unfolding of the zinc-binding domain. Collectively, structural insights into the inactive and active conformations, including wild-type and mutant proteins, suggest that the dynamic interactions of the N-terminal segments with their contacting counterpart, the interdomain linker stretch, in the reduced, inactive state are the structural determinants regulating the activation process of the post-translationally regulated chaperone, Hsp33.

Verification of the interdomain contact site in the inactive monomer, and the domain-swapped fold in the active dimer of Hsp33 in solution.

Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain-swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.

De novo generation of short antimicrobial peptides with simple amino acid composition.

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L5K2W2), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L4K5W2) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.

HP0902 from Helicobacter pylori is a thermostable, dimeric protein belonging to an all-beta topology of the cupin superfamily.

Here, we report the first biochemical and structural characterization of the hypothetical protein HP0902 from Helicobacter pylori, in terms of structural genomics. Gel-permeation chromatography and dynamic light scattering indicated that the protein behaves as a dimer in solution. Circular dichroism spectroscopy showed that HP0902 primarily adopts a beta-structure and the protein was highly thermostable with a denaturing temperature higher than 70 degrees C. Finally, the backbone NMR assignments were obtained on the [(13)C,(15)N]HP0902 and the secondary structure was determined using the chemical shift data. Additionally, the local flexibility was assessed via a heteronuclear (1)H-(15)N steady state NOE experiment. The results revealed that HP0902 would adopt a compactly folded, all-beta topology with 11 beta-strands. All of the results clearly support the notion that HP0902 belongs to the cupin superfamily of proteins.