Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality

Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.

Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin

Conformational polymorphism of m7GTP in crystal structure of the PB2 middle domain from human influenza A virus.

Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m(7)GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m(7)GTP at 1.9 Å and 2.0 Å resolution, respectively, which has two mutations (P453H, I471T) to increase electrostatic potential and solubility. Here we report the m(7)GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m(7)GTP may be a clue for the anti-influenza drug-design

B-factor plot for Cα carbons in m⁷GTP-bound structures.

<p>A) B-factor of the structure in this report is plotted in green, 2VQZ is in magenta, 4EQK is in cyan and 4ES5 is in orange. Active site residues are indicated by vertical lines and labeled in black. B) Mean B-factor values of whole molecule without m⁷GTP (cyan), m⁷GTP only (magenta) and active site residues bound to m⁷GTP (green). C) Changes of B-factor values of the active site residues between apo (blue) and m⁷GTP (red).</p

NADase activities of CPILE-a and Ia.

<p><b>A,</b> 37°C one hour incubation <b>B,</b> 25°C overnight incubation. ADP-ribose and nicotinamide eluted at 1.6 min and 4.0 min, respectively.</p

Electrostatic surfaces of the PB2 middle domain.

<p>A) Apo form and B) m⁷GTP-bound form in H1N1 and C) m⁷GTP-bound form in H3N2. The molecular orientation is the same as bottom view in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082020#pone-0082020-g001" target="_blank">Figure 1 and Figure 3</a>. Yellow arrows show the channels, which might bind the continuous RNA. Electrostatic surfaces were calculated and displayed using PyMOL.</p

Crystal structure of PB2 middle domain (amino acids 318 to 483) of H1N1 from human influenza A virus with or without m⁷GTP.

<p>A) Crystal structure without m⁷GTP in magenta. Left panel is the overall structure. Right panel is the same model as left panel but rotated by 60° about a horizontal axis to show the active site. Secondary structures are labeled in black and two mutations P453H and I471T are indicated by arrows with blue and red labels, respectively. B) Close-up view of the square of panel A in stereo view. Main chain is represented by white ribbon. Residues of active site and N-terminal helix are labeled in black. C) Crystal structure with m⁷GTP in green. Left panel is the overall structure. Right panel is the same model as left panel but rotated by 60° about a horizontal axis to show the active site. m⁷GTP is represented by stick model. Secondary structures are labeled in black and two mutations P453H and I471T are indicated by arrows with blue and red labels, respectively. D) Close-up view of the square of panel of C in stereo view. Main chain is represented by white ribbon. Active site residues are labeled in black. H357, F404, E361 and K376 formed the active site cleft to bind to 7-methyl-guanine. Triphosphate interacts with R332, S337, K339, R355 and E361.</p