Structural insights into novel mechanisms of inhibition of the major ß-carbonic anhydrase CafB from the pathogenic fungus Aspergillus fumigatus.

In fungi the ß-class of carbonic anhydrases (ß-CAs) are zinc metalloenzymes that are essential for growth, survival, differentiation, and virulence. Aspergillus fumigatus is the most important pathogen responsible for invasive aspergillosis and possesses two major ß-CAs, CafA and CafB. Recently we reported the biochemical characterization and 1.8 Å crystal structure of CafA. Here, we report a crystallographic analysis of CafB revealing the mechanism of enzyme catalysis and establish the relationship of this enzyme to other ß-CAs. While CafA has a typical open conformation, CafB, when exposed to acidic pH and/or an oxidative environment, has a novel type of active site in which a disulfide bond is formed between two zinc-ligating cysteines, expelling the zinc ion and stabilizing the inactive form of the enzyme. Based on the structural data, we generated an oxidation-resistant mutant (Y159A) of CafB. The crystal structure of the mutant under reducing conditions retains a catalytic zinc at the expected position, tetrahedrally coordinated by three residues (C57, H113 and C116) and an aspartic acid (D59), and replacing the zinc-bound water molecule in the closed form. Furthermore, the active site of CafB crystals grown under zinc-limiting conditions has a novel conformation in which the solvent-exposed catalytic cysteine (C116) is flipped out of the metal coordination sphere, facilitating release of the zinc ion. Taken together, our results suggest that A. fumigatus use sophisticated activity-inhibiting strategies to enhance its survival during infection.

The structural basis of the low catalytic activities of the two minor ß-carbonic anhydrases of the filamentous fungus Aspergillus fumigatus.

The ß-carbonic anhydrases (ß-CAs) are widely distributed zinc-metalloenzymes that play essential roles in growth, survival, development and virulence in fungi. The majority of filamentous ascomycetes possess multiple ß-CA isoforms among which major and minor forms have been characterized. We examined the catalytic behavior of the two minor ß-CAs, CafC and CafD, of Aspergillus fumigatus, and found that both enzymes exhibited low CO2 hydration activities. To understand the structural basis of their low activities, we performed X-ray crystallographic and site-directed mutagenesis studies. Both enzymes exist as homodimers. Like other Type-I ß-CAs, the CafC active site has an "open" conformation in which the zinc ion is tetrahedrally coordinated by three residues (C36, H88 and C91) and a water molecule. However, L25 and L78 on the rim of the catalytic entry site protrude into the active site cleft, partially occluding access to it. Single (L25G or L78G) and double mutants provided evidence that widening the entrance to the active site greatly accelerates catalytic activity. By contrast, CafD has a typical Type-II "closed" conformation in which the zinc-bound water molecule is replaced by aspartic acid (D36). The most likely explanation for this result is that an arginine that is largely conserved within the ß-CA family is replaced by glycine (G38), so that D36 cannot undergo a conformational change by forming a D-R pair that creates the space for a zinc-bound water molecule and switches the enzyme to the active form. The CafD structure also reveals the presence of a "non-catalytic" zinc ion in the dimer interface, which may contribute to stabilizing the dimeric assembly.

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metalfree porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Avenanthramide-C Restores Impaired Plasticity and Cognition in Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive decline and dementia with no effective treatment. Here, we investigated a novel compound from oats named avenanthramide-C (Avn-C), on AD-related memory impairment and behavioral deficits in transgenic mouse models. Acute hippocampal slices of wild-type or AD transgenic mice were treated with Avn-C in the presence or absence of oligomeric Aß42. LTP analyses and immunoblotting were performed to assess the effect of Avn-C on Aß-induced memory impairment. To further investigate the effect of Avn-C on impaired memory and Aß pathology, two different AD transgenic mice (Tg2576 and 5XFAD) models were orally treated with either Avn-C or vehicle for 2 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. Avn-C reversed impaired LTP in both ex vivo- and in vivo-treated AD mice hippocampus. Oral administration (6 mg/kg per day) for 2 weeks in AD mice leads to improved recognition and spatial memory, reduced caspase-3 cleavage, reversed neuroinflammation, and to accelerated glycogen synthase kinase-3ß (pS9GSK-3ß) and interleukin (IL-10) levels. Avn-C exerts its beneficial effects by binding to α1A adrenergic receptors to stimulate adenosine monophosphate-activated kinase (AMPK). All of the beneficial effects of Avn-C on LTP retrieval could be blocked by prazosin hydrochloride, a specific inhibitor of α1A adrenergic receptors. Our findings provide evidence, for the first time, that oats' Avn-C reverses the AD-related memory and behavioral impairments, and establish it as a potential candidate for Alzheimer's disease drug development.

Estrogen deficiency increases variability of tissue mineral density of alveolar bone surrounding teeth.

OBJECTIVE: Estrogen deficiency increases bone remodeling leading to increased variability of tissue mineral density (TMD). Due to the functional demands of mastication, alveolar bone around teeth is inherently a highly remodeled region of bone tissue with a highly variable distribution of TMD. This study investigated the effect of estrogen deficiency on the TMD distribution of alveolar bone. DESIGN: Using three-dimensional micro-computed tomography images of sham surgery (Sham) and ovariectomized (OVX) rat mandible sections, alveolar bone region (AB) and control bone region (CB) of interest were isolated. Based on histograms of gray levels equivalent to TMD values, mean (Mean), standard deviation (SD) and coefficient of variation (COV=SD/Mean) were computed. Fifth and 95th percentile gray level values were also obtained (Low(5) and High(5), respectively). Absolute value of percentage (%) differences of the gray level parameters between AB and CB regions were computed. RESULTS: Both SD and COV were significantly higher in AB region than those in CB region for all specimens of both Sham and OVX groups (p<0.001). The mean values of % differences for SD were moderately higher (p<0.073) and those for COV and Low(5) were significantly higher for the OVX group than for the Sham group (p<0.04). CONCLUSIONS: Higher variability of mineralization observed in AB of OVX group indicates that estrogen deficiency amplifies the active bone remodeling of AB already present due to the mastication. These findings provide an insight that the increased variability of TMD induced by estrogen deficiency may compromise the mechanical stability of the tooth-bearing alveolar bone.