Chia I. Liu

A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence

Staphylococcus aureus produces hospital- and community-acquired infections, with methicillin-resistant S. aureus posing a serious public health threat. The golden carotenoid pigment of S. aureus, staphyloxanthin, promotes resistance to reactive oxygen species and host neutrophil-based killing, and early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. We determined the crystal structures of S. aureus dehydrosqualene synthase (CrtM) at 1.58 angstrom resolution, finding structural similarity to human squalene synthase (SQS). We screened nine SQS inhibitors and determined the structures of three, bound to CrtM. One, previously tested for cholesterol-lowering activity in humans, blocked staphyloxanthin biosynthesis in vitro (median inhibitory concentration ∼100 nM), resulting in colorless bacteria with increased susceptibility to killing by human blood and to innate immune clearance in a mouse infection model. This finding represents proof of principle for a virulence factor–based therapy against S. aureus.

Structural and functional analysis of three β-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis

β-glucosidases (EC 3.2.1.21) cleave β-glucosidic linkages in disaccharide or glucose-substituted molecules and play important roles in fundamental biological processes. β-Glucosidases have been widely used in agricultural, biotechnological, industrial and medical applications. In this study, a high yield expression (70-250 mg/l) in Escherichia coli of the three functional β-glucosidase genes was obtained from the bacterium Clostridium cellulovorans (CcBglA), the fungus Trichoderma reesei (TrBgl2), and the termite Neotermes koshunensis (NkBgl) with the crystal structures of CcBglA, TrBgl2 and NkBgl, determined at 1.9Å, 1.63Å and 1.34Å resolution, respectively. The overall structures of these enzymes are similar to those belonging to the β-retaining glycosyl hydrolase family 1, which have a classical (α/β)(8)-TIM barrel fold. Each contains a slot-like active site cleft and a more variable outer opening, related to its function in processing different lengths of β-1,4-linked glucose derivatives. The two essential glutamate residues for hydrolysis are spatially conserved in the active site. In both TrBgl2 and NkBgl structures, a Tris molecule was found to bind at the active site, explaining the slight inhibition of hydrolase activity observed in Tris buffer. Manganese ions at 10mM exerted an approximate 2-fold enzyme activity enhancement of all three β-glucosidases, with CcBglA catalyzing the most efficiently in hydrolysis reaction and tolerating Tris as well as some metal inhibition. In summary, our results for the structural and functional properties of these three β-glucosidases from various biological sources open important avenues of exploration for further practical applications.

Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylococcus aureus: in vitro, in vivo, and crystallographic results.

The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis inhibitors that inhibit the first committed step in its biosynthesis, condensation of two farnesyl diphosphate (FPP) molecules to dehydrosqualene, catalyzed by the enzyme dehydrosqualene synthase (CrtM). The most active compounds are phosphonoacetamides that have low nanomolar K(i) values for CrtM inhibition and are active in whole bacterial cells and in mice, where they inhibit S. aureus disease progression. We also report the X-ray crystallographic structure of the most active compound, N-3-(3-phenoxyphenyl)propylphosphonoacetamide (IC(50) = 8 nM, in cells), bound to CrtM. The structure exhibits a complex network of hydrogen bonds between the polar headgroup and the protein, while the 3-phenoxyphenyl side chain is located in a hydrophobic pocket previously reported to bind farnesyl thiodiphosphate (FsPP), as well as biphenyl phosphonosulfonate inhibitors. Given the good enzymatic, whole cell, and in vivo pharmacologic activities, these results should help guide the further development of novel antivirulence factor-based therapies for S. aureus infections.

Mechanism of action and inhibition of dehydrosqualene synthase

“Head-to-head” terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg2+ cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis

Expression of the gene cluster icaADBC is necessary for biofilm production in Staphylococcus epidermidis. The ica operon is negatively controlled by the repressor IcaR. Here, the crystal structure of IcaR was determined and the refined structure revealed a homodimer comprising entirely α-helices, typical of the tetracycline repressor protein family for gene regulations. The N-terminal domain contains a conserved helix-turn-helix DNA-binding motif with some conformational variations, indicating flexibility in this region. The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation. The results of the electrophoretic mobility shift assay and isothermal titration calorimetry suggested that a 28 bp core segment of the ica operator is implicated in the cooperative binding of two IcaR dimers on opposite sides of the duplex DNA. Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein–DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences. By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

Binding modes of zaragozic acid A to human squalene synthase and staphylococcal dehydrosqualene synthase

Background: Fungal metabolites, zaragozic acids, are potent inhibitors against squalene synthase and S. aureus dehydro-squalene synthase. Results: Here we reported the zaragozic acid A binding mode for the head-to-head prenyltransferase drug targets. Conclusion: Zaragozic acid A presents an unexpected binding mode from the cyclopropyl-containing intermediate in both enzymes. Significance: Our structures provide a structural basis for development of potential drugs for hyperlipidemias and bacterial infection treatments. Zaragozic acids (ZAs) belong to a family of fungal metabolites with nanomolar inhibitory activity toward squalene synthase (SQS). The enzyme catalyzes the committed step of sterol synthesis and has attracted attention as a potential target for antilipogenic and antiinfective therapies. Here, we have determined the structure of ZA-A complexed with human SQS. ZA-A binding induces a local conformational change in the substrate binding site, and its C-6 acyl group also extends over to the cofactor binding cavity. In addition, ZA-A effectively inhibits a homologous bacterial enzyme, dehydrosqualene synthase (CrtM), which synthesizes the precursor of staphyloxanthin in Staphylococcus aureus to cope with oxidative stress. Size reduction at Tyr248 in CrtM further increases the ZA-A binding affinity, and it reveals a similar overall inhibitor binding mode to that of human SQS/ZA-A except for the C-6 acyl group. These structures pave the way for further improving selectivity and development of a new generation of anticholesterolemic and antimicrobial inhibitors.

Crowning Proteins: Modulating the Protein Surface Properties using Crown Ethers.

Crown ethers are small, cyclic polyethers that have found wide-spread use in phase-transfer catalysis and, to a certain degree, in protein chemistry. Crown ethers readily bind metallic and organic cations, including positively charged amino acid side chains. We elucidated the crystal structures of several protein-crown ether co-crystals grown in the presence of 18-crown-6. We then employed biophysical methods and molecular dynamics simulations to compare these complexes with the corresponding apoproteins and with similar complexes with ring-shaped low-molecular-weight polyethylene glycols. Our studies show that crown ethers can modify protein surface behavior dramatically by stabilizing either intra- or intermolecular interactions. Consequently, we propose that crown ethers can be used to modulate a wide variety of protein surface behaviors, such as oligomerization, domain–domain interactions, stabilization in organic solvents, and crystallization.

High-resolution structures of Neotermes koshunensis β-glucosidase mutants provide insights into the catalytic mechanism and the synthesis of glucoconjugates.

NkBgl, a β-glucosidase from Neotermes koshunensis, is a β-retaining glycosyl hydrolase family 1 enzyme that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. β-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in β-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve β-glucosidases for the enzymatic synthesis of oligosaccharides.

Structures of human MST3 kinase in complex with adenine, ADP and Mn2+.

The MST family is a subclass of mammalian serine/threonine kinases that are related to the yeast sterile-20 protein and are implicated in regulating cell growth and transformation. The MST3 protein contains a 300-residue catalytic domain and a 130-residue regulatory domain, which can be cleaved by caspase and activated by autophosphorylation, promoting apoptosis. Here, five crystal structures of the catalytic domain of MST3 are presented, including a complex with ADP and manganese, a unique cofactor preferred by the enzyme, and a complex with adenine. Similar to other protein kinases, the catalytic domain of MST3 folds into two lobes: the smaller N lobe forms the nucleotide-binding site and the larger C lobe recognizes the polypeptide substrate. The bound ADP and Mn(2+) ions are covered by a glycine-rich loop and held in place by Asn149 and Asp162. A different orientation was observed for the ligand in the MST3-adenine complex. In the activation loop, the side chain of Thr178 is phosphorylated and is sandwiched by Arg143 and Arg176. Comparison of this structure with other similar kinase structures shows a 180 degrees rotation of the loop, leading to activation of the enzyme. The well defined protein-ligand interactions also provide useful information for the design of potent inhibitors.

Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum

Non‐mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor‐growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino‐terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA– cells was associated with the impairment of phagocytosis and fluid‐phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.