Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study, we characterize a 120-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho family GTPases, Rac1, Rnd1, and RhoD. The NMR data show that, surprisingly, the Cdc42/Rac interactive binding-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, ␤-strands 3 and 4 and a short ␣-helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a four-stranded antiparallel ␤-sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.Members of the plexin family of transmembrane receptors have important functions in guiding axon growth in the developing nervous system (1-3). Plexins also function in several developmental processes such as cardiovascular development and angiogenesis (4 -8), in the invasive growth of epithelial cells (9), and in the immune system (10). The extracellular part of plexin shares significant homology with that of the hepatocyte growth factor receptor as well as with semaphorins, the principal family of ligands for plexins. The cytoplasmic region of plexins is also well conserved (11). Two segments with homology to Ras GTPase-activating proteins (GAPs) 5 are linked by a region that has been identified as the location for the binding of several Rho family GTPases.Plexins are unique in that they are the first documented example of a transmembrane receptor that interacts directly with small GTPases. Most A-and B-family plexins bind activated Rac1 and Rnd1 (12-15), Rho family GTPases that are known regulators of cytoskeletal dynamics and cell adhesion (16). Plexin-A1 has also been shown to bind active RhoD (17), another Rho family GTPase involved in actin remodeling and endosomal dynamics and possibly in receptor down-regulation (18). The role of these plexin-Rho GTPase interactions has remained unclear, however, as have the characteristics of the binding region. Does the Rho GTPase binding segment provide a GTPase regulatory property, functioning akin to a guanine nucleotide dissociation inhibitor by sequestering certain Rho family GTPases, or does it act as an effector pro...

Sterol 14␣-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.