Lysophosphatidic acids (LPAs) are ubiquitous serum phospholipids that trigger diverse cellular responses such as cell proliferation, migration, cell survival, and calcium influx. LPA receptors belong to the G‐protein coupled receptor (GPCR) family, and seven subtypes of LPA receptors have been identified. Among them, LPA receptor 2 (LPA 2 ) is involved in the proliferation and metastasis of ovarian, cervical, and breast cancers. Hence, LPA 2 ‐specific antagonists or antibodies are considered as potential anticancer therapeutics. To develop antibodies against LPA 2 , a recombinant LPA 2 was expressed in Escherichia coli , purified to homogeneity, and immobilized on a solid surface as an active conformation. An M13 phage library displaying single‐chain variable fragments (scFvs) of human IgG containing randomized complementarity‐determining regions was applied to select LPA 2 ‐specific scFv clones. After five rounds of biopanning, a few scFv clones which showed specific binding to LPA 2 were identified. Single‐chain antibodies (scAbs), which contain the isolated scFvs and Cκ domain, were constructed and expressed in E. coli . The purified scAbs showed specific binding to LPA 2 with K D values of 200–600 nM.

DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

Bacterial cytokinesis is accomplished by the Z-ring, which is a polymeric structure that includes the tubulin homologue FtsZ at the division site. ZapD, a Z-ring-associated protein, directly binds to FtsZ and stabilizes the polymerization of FtsZ to form a stable Z-ring during cytokinesis. Structural analysis of ZapD from Escherichia coli was performed to investigate the mechanism of ZapD-mediated FtsZ stabilization and polymerization. ZapD was crystallized using a reservoir solution consisting of 1.5 M lithium sulfate, 0.1 M HEPES pH 7.8, 2%(v/v) polyethylene glycol 400. X-ray diffraction data were collected to 2.95 Å resolution. The crystals belonged to the hexagonal space group P64, with unit-cell parameters a = b = 109.5, c = 106.7 Å, γ = 120.0°. Two monomers were present in the asymmetric unit, resulting in a crystal volume per protein mass (VM) of 3.25 Å(3) Da(-1) and a solvent content of 62.17%.

CO2 fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase from Candida boidinii (CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations, i.e. open (NAD(+)-unbound) and closed (NAD(+)-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2 reduction than in CbFDH.

Gli3 interact with SPOP. AUC supports a binding model in which the MATH domain can interact with three binding motifs on Gli3-90 with macroscopic weak affinities while FA allows the determination of microscopic affinities. In vitro ubiquitination assays and protein stability studies in mammalian cells with a series of Gli3 SBCmotif mutants are used for functional interpretation of our biophysical data. Each SBC motif contributes to SPOP binding in agreement with their similar weak affinities. These results will allow the distinction between a dynamic complex, a static multivalent binding mechanism and large oligomeric assemblies in which many Gli3s co-operate to recruit multiple SPOP dimers. The presence of multiple dispersed SBC motifs suggests their contribution to spatial and temporal control of Gli3 levels in response to Hh signaling.

Bacterial cytokinesis is accompanied by a macro-molecular complex called the "divisome." The divisome consists of two major components involving positive regulators and negative regulators that regulate the polymerization of an essential cytoskeleton protein FtsZ, which plays a key role in bacterial cell division by assembling the Z-ring, and therefore has been identified as a target for antibiotics. The negative regulators prevent the Z-ring assembly by inhibiting FtsZ polymerization. In Staphylococcus aureus, a pandemic human pathogen, one of the negative regulators, EzrA, contains a trans-membrane anchor region at the N-terminus and has five predicted coiled-coils. Recent reports indicate that the polymerization of FtsZ can be inhibited by forming a complex with EzrA. In this study, we attempted to locate the binding site for the interaction between EzrA and FtsZ in S. aureus (SaEzrA and SaFtsZ, respectively), by generating various constructs of SaEzrA and SaFtsZ proteins based on limited proteolysis. Various constructs of SaEzrA and SaFtsZ proteins were expressed and homogeneously purified. A GST pull-down assay indicated that the N-terminal domain of SaEzrA interacts with the C-terminal tail of SaFtsZ, and the elongated shape of EzrA was predicted based on the Stokes radius of each construct.

Significance Structural and functional studies on protein kinase CK2α, which is a ubiquitous kinase that can phosphorylate hundreds of cellular proteins, revealed that CK2α activity is inhibited by Nopp140 and reactivated by IP 6 by competitive binding at the substrate recognition site of CK2α. IP 6 binds to the lysine-rich cluster of CK2α, and phospho-Ser574 on Nopp140 significantly enhances its interaction with CK2α.

Cytokinesis is the final stage of cell division, dividing one mother cell into two daughter cells. For the cutting of a plasma membrane during bacterial cytokinesis, a tubulin homolog FtsZ protein is recruited from the cytoplasm to the division site. FtsZ protein polymerizes in a GTP-dependent manner and its N-terminal domain has a GTPase activity. In this study, we have begun to characterize FtsZ from Staphylococcus aureus (SA). Full-length SA FtsZ was cloned into pRSFDuet-1 vector and the clone was transformed into a BL21 (DE3) star cell. The recombinant SA FtsZ protein was purified using Ni-NTA affinity chromatography and dialysis. Using a spectrofluorometer, we showed that SA FtsZ undergoes a GTP-dependant polymerization in vitro. The polymer of the SA FtsZ protein disappeared after a few minutes, suggesting that the polymer is degraded as the GTP is consumed. This assay system may well be applied for inhibitor screening targeting S. aureus FtsZ.