Correction: Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity.

[This corrects the article DOI: 10.1371/journal.pone.0152611.].

Correction: Crystal Structure of Pim1 Kinase in Complex with a Pyrido[4,3-D]Pyrimidine Derivative Suggests a Unique Binding Mode.

[This corrects the article DOI: 10.1371/journal.pone.0070358.].

Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity.

Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of a putative xylose isomerase from Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron BT0793, a putative xylose isomerase, was overexpressed in Escherichia coli, purified and crystallized using polyethylene glycol monomethyl ether 550 as the precipitant. X-ray diffraction data were collected to 2.10 Šresolution at 100 K using synchrotron X-rays. The crystal was found to belong to space group P1, with unit-cell parameters a=96.3, b=101.7, c=108.3 Å, α=82.8, ß=68.2, γ=83.0°. The asymmetric unit contained eight subunits of xylose isomerase with a crystal volume per protein weight (VM) of 2.38 Å3 Da(-1) and a solvent content of 48.3%.

Crystal structure of pim1 kinase in complex with a pyrido[4,3-d]pyrimidine derivative suggests a unique binding mode.

Human Pim1 kinase is a serine/threonine protein kinase that plays important biological roles in cell survival, apoptosis, proliferation, and differentiation. Moreover, Pim1 is up-regulated in various hematopoietic malignancies and solid tumors. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small molecule inhibitors for Pim1. Here, we describe the crystal structure of Pim1 in complex with a newly developed pyrido[4,3-d]pyrimidine-derivative inhibitor (SKI-O-068). Our inhibitor exhibits a half maximum inhibitory concentration (IC50) of 123 (±14) nM and has an unusual binding mode in complex with Pim1 kinase. The interactions between SKI-O-068 and the Pim1 active site pocket residue are different from those of other scaffold inhibitor-bound structures. The binding mode analysis suggests that the SKI-O-068 inhibitor can be improved by introducing functional groups that facilitate direct interaction with Lys67, which aid in the design of an optimized inhibitor.

Crystal structure of Pyrococcus furiosus PF2050, a member of the DUF2666 protein family.

Pyrococcus furiosus PF2050 is an uncharacterized putative protein that contains two DUF2666 domains. Functional and structural studies of PF2050 have not previously been performed. In this study, we determined the crystal structure of PF2050. The structure of PF2050 showed that the two DUF2666 domains interact tightly, forming a globular structure. Each DUF2666 domain comprises an antiparallel ß-sheet and an α-helical bundle. One side of the PF2050 structure has a positively charged basic cleft, which may have a DNA-binding function. Furthermore, we confirmed that PF2050 interacts with circular and linear dsDNA.

Helical repeat structure of apoptosis inhibitor 5 reveals protein-protein interaction modules.

Apoptosis inhibitor 5 (API5) is an anti-apoptotic protein that is up-regulated in various cancer cells. Here, we present the crystal structure of human API5. API5 exhibits an elongated all α-helical structure. The N-terminal half of API5 is similar to the HEAT repeat and the C-terminal half is similar to the ARM (Armadillo-like) repeat. HEAT and ARM repeats have been implicated in protein-protein interactions, suggesting that the cellular roles of API5 may be to mediate protein-protein interactions. Various components of multiprotein complexes have been identified as API5-interacting protein partners, suggesting that API5 may act as a scaffold for multiprotein complexes. API5 exists as a monomer, and the functionally important heptad leucine repeat does not exhibit the predicted a dimeric leucine zipper. Additionally, Lys-251, which can be acetylated in cells, plays important roles in the inhibition of apoptosis under serum deprivation conditions. The acetylation of this lysine also affects the stability of API5 in cells.

Crystal structure of human transglutaminase 2 in complex with adenosine triphosphate.

Transglutaminase 2 (TG2) is a calcium-dependent multifunctional protein associated with various human diseases. We determined the crystal structure of human TG2 in complex with adenosine triphosphate (ATP). The ATP molecule binds to the previously identified guanosine diphosphate (GDP) binding pocket but has different hydrogen bonds and ion interaction with protein. The four residues Arg476, Arg478, Val479 and Tyr583, all of which are involved in both ATP and GDP binding by hydrogen bonds, might play important roles in the stabilization of TG2 by ATP or GDP. However, Ser482 and Arg580, which are involved in GDP binding, do not form hydrogen bond with ATP. Additionally, we newly discovered an intramolecular disulfide bond between Cys230 and Cys370, which formation might regulate the enzymatic activity of TG2.

Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins.

Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1-134) and HRASLS3 (1-133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A(2) activities. Limited proteolysis revealed that TIG3 (1-132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.

Differential cytotoxic effects of mono-(2-ethylhexyl) phthalate on blastomere-derived embryonic stem cells and differentiating neurons.

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 microM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 microM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.