Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy.

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

Structural insights into adiponectin receptors suggest ceramidase activity.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

Systematic bioinformatics and experimental validation of yeast complexes reduces the rate of attrition during structural investigations.

For high-throughput structural studies of protein complexes of composition inferred from proteomics data, it is crucial that candidate complexes are selected accurately. Herein, we exemplify a procedure that combines a bioinformatics tool for complex selection with in vivo validation, to deliver structural results in a medium-throughout manner. We have selected a set of 20 yeast complexes, which were predicted to be feasible by either an automated bioinformatics algorithm, by manual inspection of primary data, or by literature searches. These complexes were validated with two straightforward and efficient biochemical assays, and heterologous expression technologies of complex components were then used to produce the complexes to assess their feasibility experimentally. Approximately one-half of the selected complexes were useful for structural studies, and we detail one particular success story. Our results underscore the importance of accurate target selection and validation in avoiding transient, unstable, or simply nonexistent complexes from the outset.

Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents.

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.