P47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X‐ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane‐binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4‐bisphosphate [PtdIns(3,4)P2] and is analogous to the phophatidylinositol 3‐phosphate (PtdIns3P)‐binding pocket of p40 phox , while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid‐binding pockets radically increases membrane affinity. Strikingly, measurements for full‐length p47 phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C‐terminal SH3 domain (C‐SH3). Either a site‐specific mutation in C‐SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

Cytokines of the gp130 family exert their diverse biological effects by formation of stable high affinity transmembrane receptor complexes that are characterized by the presence of the shared transmembrane signalling receptor gp130. Different gp130 ligands form signalling complexes that vary in both composition and stoichiometry. Analysis of the three‐dimensional structure of selected ligands and receptor elements indicates that ligands display three topologically conserved receptor recognition epitopes that interact with complementary ligand recognition elements. The composition of the signalling complex and downstream biological responses is defined by the relative affinity of different receptor components for these epitopes. The detailed structure of receptor recognition epitopes indicates that the generation of small molecule cytokine mimetics may be a feasible objective.