Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50.

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S. aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S. aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Functional details of the Mycobacterium tuberculosis VapBC26 toxin-antitoxin system based on a structural study: insights into unique binding and antibiotic peptides.

Toxin-antitoxin (TA) systems are essential for bacterial persistence under stressful conditions. In particular, Mycobacterium tuberculosis express VapBC TA genes that encode the stable VapC toxin and the labile VapB antitoxin. Under normal conditions, these proteins interact to form a non-toxic TA complex, but the toxin is activated by release from the antitoxin in response to unfavorable conditions. Here, we present the crystal structure of the M. tuberculosis VapBC26 complex and show that the VapC26 toxin contains a pilus retraction protein (PilT) N-terminal (PIN) domain that is essential for ribonuclease activity and that, the VapB26 antitoxin folds into a ribbon-helix-helix DNA-binding motif at the N-terminus. The active site of VapC26 is sterically blocked by the flexible C-terminal region of VapB26. The C-terminal region of free VapB26 adopts an unfolded conformation but forms a helix upon binding to VapC26. The results of RNase activity assays show that Mg2+ and Mn2+ are essential for the ribonuclease activity of VapC26. As shown in the nuclear magnetic resonance spectra, several residues of VapB26 participate in the specific binding to the promoter region of the VapBC26 operon. In addition, toxin-mimicking peptides were designed that inhibit TA complex formation and thereby increase toxin activity, providing a novel approach to the development of new antibiotics.

Identification of novel scaffolds for potential anti-Helicobacter pylori agents based on the crystal structure of H. pylori 3-deoxy-d-manno-octulosonate 8-phosphate synthase (HpKDO8PS).

The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDO8PS) from Helicobacter pylori (HpKDO8PS) was determined alone and within various complexes, revealing an extra helix (HE) that is absent in the structures of KDO8PS from other organisms. In contrast to the metal coordination of the KDO8PS enzyme from Aquifex aeolicus, HpKDO8PS is specifically coordinated with Cd(2+) or Zn(2+) ions, and isothermal titration calorimetry (ITC) and differential scanning fluorimetry (DSF) revealed that Cd(2+) thermally stabilizes the protein structure more efficiently than Zn(2+). In the substrate-bound structure, water molecules play a key role in fixing residues in the proper configuration to achieve a compact structure. Using the structures of HpKDO8PS and API [arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) bisubstrate inhibitor], we generated 21 compounds showing potential HpKDO8PS-binding properties via in silico virtual screening. The capacity of three, avicularin, hyperin, and MC181, to bind to HpKDO8PS was confirmed through saturation transfer difference (STD) experiments, and we identified their specific ligand binding modes by combining competition experiments and docking simulation analysis. Hyperin was confirmed to bind to the A5P binding site, primarily via hydrophilic interaction, whereas MC181 bound to both the PEP and A5P binding sites through hydrophilic and hydrophobic interactions. These results were consistent with the epitope mapping by STD. Our results are expected to provide clues for the development of HpKDO8PS inhibitors.

Alba from Thermoplasma volcanium belongs to α-NAT's: An insight into the structural aspects of Tv Alba and its acetylation by Tv Ard1.

The Alba superfamily proteins have been regarded as a conserved group of proteins in archaea and eukarya, which have shown to be important in nucleic acid binding, chromatic organization and gene regulation. These proteins often belong to the N-acetyltransferase (NAT) category (N(α)-acetyltransferases or N(ε)-acetyltransferases) and undergo post-translational modifications. Here, we report the crystal structure of Alba from Thermoplasma volcanium (Tv Alba) at 2.4 Å resolution. The acetylation of Tv Alba was monitored and the N-terminal of Tv Alba has been shown to interact with acetyl coenzyme A (Ac-CoA). The chemical shift perturbation experiments of Tv Alba were performed in the presence of Ac-CoA and/or Tv Ard1, another T. volcanium protein that treats Tv Alba as a substrate. To examine the DNA binding capabilities of Tv Alba alone and in the presence of Ac-CoA and/or Tv Ard1, EMSA experiments were carried out. It is shown that although Tv Alba binds to Ac-CoA, the acetylation of Tv Alba is not related with its binding to dsDNA, and the involvement of the N-terminus in Ac-CoA binding demonstrates that Tv Alba belongs to the N(α)-acetyltransferase family.

ß-Arm flexibility of HU from Staphylococcus aureus dictates the DNA-binding and recognition mechanism.

HU, one of the major nucleoid-associated proteins, interacts with the minor groove of DNA in a nonspecific manner to induce DNA bending or to stabilize bent DNA. In this study, crystal structures are reported for both free HU from Staphylococcus aureus Mu50 (SHU) and SHU bound to 21-mer dsDNA. The structures, in combination with electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC) measurements and molecular-dynamics (MD) simulations, elucidate the overall and residue-specific changes in SHU upon recognizing and binding to DNA. Firstly, structural comparison showed the flexible nature of the ß-sheets of the DNA-binding domain and that the ß-arms bend inwards upon complex formation, whereas the other portions are nearly unaltered. Secondly, it was found that the disruption and formation of salt bridges accompanies DNA binding. Thirdly, residue-specific free-energy analyses using the MM-PBSA method with MD simulation data suggested that the successive basic residues in the ß-arms play a central role in recognizing and binding to DNA, which was confirmed by the EMSA and ITC analyses. Moreover, residue Arg55 resides in the hinge region of the flexible ß-arms, exhibiting a remarkable role in their flexible nature. Fourthly, EMSAs with various DNAs revealed that SHU prefers deformable DNA. Taken together, these data suggest residue-specific roles in local shape and base readouts, which are primarily mediated by the flexible ß-arms consisting of residues 50-80.

Structure of Thermoplasma volcanium Ard1 belongs to N-acetyltransferase family member suggesting multiple ligand binding modes with acetyl coenzyme A and coenzyme A.

Acetylation and deacetylation reactions result in biologically important modifications that are involved in normal cell function and cancer development. These reactions, carried out by protein acetyltransferase enzymes, act by transferring an acetyl group from acetyl-coenzymeA (Ac-CoA) to various substrate proteins. Such protein acetylation remains poorly understood in Archaea, and has been only partially described. Information processing in Archaea has been reported to be similar to that in eukaryotes and distinct from the equivalent bacterial processes. The human N-acetyltransferase Ard1 (hArd1) is one of the acetyltransferases that has been found to be overexpressed in various cancer cells and tissues, and knockout of the hArd1 gene significantly reduces growth rate of the cancer cell lines. In the present study, we determined the crystal structure of Thermoplasma volcanium Ard1 (Tv Ard1), which shows both ligand-free and multiple ligand-bound forms, i.e.,Ac-CoA- and coenzyme A (CoA)-bound forms. The difference between ligand-free and ligand-bound chains in the crystal structure was used to search for the interacting residues. The re-orientation and position of the loop between ß4 and α3 including the phosphate-binding loop (P-loop) were observed, which are important for the ligand interaction. In addition, a biochemical assay to determine the N-acetyltransferase activity of Tv Ard1 was performed using the T.volcanium substrate protein Alba (Tv Alba). Taken together, the findings of this study elucidate ligand-free form of Tv Ard1 for the first time and suggest multiple modes of binding with Ac-CoA and CoA.

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.

Crystal structure of toxin HP0892 from Helicobacter pylori with two Zn(II) at 1.8 Å resolution.

Antibiotic resistance and microorganism virulence have been consistently exhibited by bacteria and archaea, which survive in conditions of environmental stress through toxin-antitoxin (TA) systems. The HP0892-HP0893 TA system is one of the two known TA systems belonging to Helicobacter pylori. The antitoxin, HP0893, binds and inhibits the HP0892 toxin and regulates the transcription of the TA operon. Here, we present the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues at the mRNase active site was shown. The involved residues, namely E58A, H86A, and H58A/ H60A, were mutated and the binding affinity was monitored by ITC studies. Through the structural difference between the apo and the metal-bound state, and using a homology modeling tool, the involvement of the metal ion in mRNase active site could be identified. The most catalytically important residue, His86, reorients itself to exhibit RNase activity. His47, Glu58, and His60 are involved in metal binding where Glu58 acts as a general base and His47 and His60 may also act as a general acid in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. Arg83 is involved in phosphate binding and stabilization of the transition state, and Phe90 is involved in base packing and substrate orientation.

Solution structure of Rv0569, potent hypoxic signal transduction protein, from Mycobacterium tuberculosis.

The latent infection is unique characteristic of Mycobacterium tuberculosis to overcome human immune response for its survival. The M. tb develops adaptation to extreme stress conditions to increase the viability, thus easily acquires drug resistance than any other bacteria and maintains a long-term infection status without any symptoms. Rv0569 is a conserved hypothetical protein that overexpresses under dormant state induced by hypoxia, starvation, and medication. To study function and structure in detail, we determined the solution structure of Rv0569 by NMR. NOE and RDC restraints were used to calculate the structure, which was further refined with AMBER. Rv0569 is composed of five antiparallel ß-sheets and one α-helix. Rv0569 shows structural similarity with its homolog Rv2302, yet there is a big difference in the orientation of C-terminal α-helix between Rv0569 and Rv2302. According to previous studies, Rv0569 might comprise a hypoxia induced operon with the Rv0570 which is located 29 bp downstream of the Rv0569 and Rv0570 plays an important role in the latent infection. From our structure and bioinformatics research, we suggest that Rv0569 contributes to signaling transduction in hypoxic condition by binding with DNA for upregulation of Rv0570 or supporting Rv0570 for binding ATP during dormancy of tuberculosis.

Crystal structure of apo and copper bound HP0894 toxin from Helicobacter pylori 26695 and insight into mRNase activity.

The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and microorganism virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. In Helicobacter pylori, only two TA systems are known thus far. Our previous studies showed that HP0894-HP0895 acts as a TA system and that HP0894 exhibits intrinsic RNase activity. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remain unclear. Therefore, in an attempt to shed some light on the mechanism behind the TA system of HP0894-HP0895, here we present the crystal structures of apo- and metal-bound H. pylori 0894 at 1.28Å and 1.89Å, respectively. Through the combined approach of structural analysis and structural homology search, the amino acids involved in mRNase active site were monitored and the reorientations of different residues were discussed in detail. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself to exhibit this type of activity, acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Lys52, Glu58, Asp64 and Arg80 have phosphate binding and specific sequence recognition. Glu58 also acts as a general base, and substrate reorientation is caused by Phe88. Based on experimental findings, a model for antitoxin binding could be suggested.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of hypothetical protein SAV0479 from Staphylococcus aureus Mu50.

SAV0479, a hypothetical protein from the Mu50 strain of methicillin- and vancomycin-resistant Staphylococcus aureus, was selected for structure and function determination as part of a structural genomics project. Here, the cloning, overexpression, purification and crystallization of SAV0479 are reported. Crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 81.48, c = 82.53 Å. Three monomers of SAV0479 are present in each asymmetric unit.

Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity.

VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin-antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design.

BACKGROUND: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens. RESULTS: Here we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = ß = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway. CONCLUSIONS: We have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.

Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE.

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-beta (Abeta) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substrate-bound human 113-kDa IDE reveals that the N- and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that Abeta is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in Vmax with only minimal changes to Km. Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae.

BACKGROUND: Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer. RESULTS: The alanine racemases gene from S. pneumoniae (alrSP) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively. CONCLUSION: We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site.

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.

Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms.

The structure of the catabolic alanine racemase, DadX, from the pathogenic bacterium Pseudomonas aeruginosa, reported here at 1.45 A resolution, is a dimer in which each monomer is comprised of two domains, an eight-stranded alpha/beta barrel containing the PLP cofactor and a second domain primarily composed of beta-strands. The geometry of each domain is very similar to that of Bacillus stearothermophilus alanine racemase, but the rotation between domains differs by about 15 degrees. This change does not alter the structure of the active site in which almost all residues superimpose well with a low rms difference of 0.86 A. Unexpectedly, the active site of DadX contains a guest substrate that is located where acetate and propionate have been observed in the Bacillus structures. It is modeled as d-lysine and oriented such that its terminal NZ atom makes a covalent bond with C4' of PLP. Since the internal aldimine bond between the protein lysine, Lys33, and C4' of PLP is also unambiguously observed, there appears to be an equilibrium between both internally and externally reacted forms. The PLP cofactor adopts two partially occupied conformational states that resemble previously reported internal and external aldimine complexes.

Characterization and crystallization of active domains of a novel luciferase from a marine dinoflagellate.

Lingulodinium polyedrum luciferase is a bioluminescent protein found in the marine dinoflagellate formerly known as Gonyaulax. It is located in organelles called scintillons that emit brief and bright flashes of light that are regulated by an endogenous circadian clock. The complete luciferase molecule has a molecular mass of 136 994 Da and contains three homologous domains, each of which is a separately active luciferase. Two of these domains, D2-LCF and D3-LCF, have been cloned, expressed and crystallized. Crystals of D2-LCF were obtained from PEG 10 000 in space group P2(1)2(1)2(1), with unit-cell parameters a = 49.1, b = 104.7, c = 180.3 A. They diffract to 2.9 A on a rotating anode. Crystals of D3-LCF were grown from PEG 2000 in space group P2(1)2(1)2(1), with unit-cell parameters a = 58.86, b = 63.98, c = 95.76 A. They diffract to 2.3 A on a rotating anode.