Twisting of the DNA-binding surface by a beta-strand-bearing proline modulates DNA gyrase activity.

DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA. The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrase-specific 'GyrA-box' motif within this domain are essential for this unique (-) supercoiling activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif. This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting a non-planar beta-pinwheel fold composed of six seemingly spirally arranged beta-sheet blades. Interestingly, structural analysis revealed that the non-planar architecture mainly stems from the tilted packing seen between blades 1 and 2, with the packing geometry likely being defined by a conserved and unusual beta-strand-bearing proline. Consequently, the GyrA-box-containing blade 1 is placed at an angled spatial position relative to the other DNA-binding blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface. Mutagenesis studies support that the proline-induced structural twist contributes directly to gyrase's (-) supercoiling activity. To our knowledge, this is the first demonstration that a beta-strand-bearing proline may impact protein function. Potential relevance of beta-strand-bearing proline to disease phenylketonuria is also noted.

Structure of the saxiphilin:saxitoxin (STX) complex reveals a convergent molecular recognition strategy for paralytic toxins.

Dinoflagelates and cyanobacteria produce saxitoxin (STX), a lethal bis-guanidinium neurotoxin causing paralytic shellfish poisoning. A number of metazoans have soluble STX-binding proteins that may prevent STX intoxication. However, their STX molecular recognition mechanisms remain unknown. Here, we present structures of saxiphilin (Sxph), a bullfrog high-affinity STX-binding protein, alone and bound to STX. The structures reveal a novel high-affinity STX-binding site built from a "proto-pocket" on a transferrin scaffold that also bears thyroglobulin domain protease inhibitor repeats. Comparison of Sxph and voltage-gated sodium channel STX-binding sites reveals a convergent toxin recognition strategy comprising a largely rigid binding site where acidic side chains and a cation-π interaction engage STX. These studies reveal molecular rules for STX recognition, outline how a toxin-binding site can be built on a naïve scaffold, and open a path to developing protein sensors for environmental STX monitoring and new biologics for STX intoxication mitigation.

Regulation of the Escherichia coli HipBA toxin-antitoxin system by proteolysis.

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.

Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide.

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2ß complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.

Inhibition of glutathione S-transferase M1 by new gabosine analogues is essential for overcoming cisplatin resistance in lung cancer cells.

A new class of human GST inhibitors has been identified via rational design approach; we report their discovery, synthesis, inhibitory activity, and synergetic effect in combination with cisplatin against A549 lung cancer cell line. The results of this effort show that the lead 4-O-decyl-gabosine D (24) has optimum synergetic effect in A549 human lung adenocarcinoma epithelial cell and that this activity involves inhibition of glutathione S-transferase M1, apparently consistent with siRNA-mediated knockdown of GSTM1 gene.