In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.

As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.

IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2R␣, IL-2R␤, and ␥c. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 Å resolution. The quaternary structure is consistent with a stepwise assembly from IL-2͞IL-2R␣ to IL-2͞IL-2R␣͞IL-2R␤ to IL-2͞IL-2R␣͞IL-2R␤͞␥c. The IL-2R␣ subunit forms the largest of the three IL-2͞IL-2R interfaces, which, together with the high abundance of charge-charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2R␣ with IL-2 at the cell surface. Surprisingly, IL-2R␣ makes no contacts with IL-2R␤ or ␥c, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2R␣ to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2R␤ and ␥c subunits. Helix A of IL-2 wedges tightly between IL-2R␤ and ␥c to form a three-way junction that coalesces into a composite binding site for the final ␥c recruitment. The IL-2͞␥c interface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.common ␥ chain ͉ cooperativity ͉ IL-2 receptor ͉ receptor assembly ͉ structure-activity relationship

We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three crystal structures of the nucleoporin pair Seh1*Nup85, which is part of the coat cylinder. The Seh1*Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13*Nup145C. Furthermore, the Seh1*Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1*Nup85 and Sec13*Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

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Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-L-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.crystal structure | enzyme catalysis | PRMT | histone methylation | epigenetics P osttranslational modifications of proteins can affect their structure, catalytic activity, and molecular interactions (1). Methylation of the guanidino group of arginine residues represents a prominent subset of these reactions (2). Histone arginine methylation is associated with gene silencing and activation (3); the modification of arginine residues in a variety of nonhistone proteins, including splicing and transcription factors, can regulate their activity (4, 5).Most of the enzymes that catalyze arginine methylation are designated protein arginine methyltransferases (PRMTs) and require the cofactor S-adenosyl-L-methionine (AdoMet) as the methyl donor (6). Four types of arginine methylation products havedimethylarginine (SDMA), and δ-N G -monomethylarginine (6, 7). Accordingly, PRMTs can be categorized into four groups: Type I PRMTs catalyze ADMA formation, type II PRMTs catalyze SDMA formation, type III PRMTs catalyze MMA formation, and type IV PRMTs catalyze δ-N G -monomethylarginine formation. Type I, II, and III PRMTs are widely distributed in nature whereas type IV PRMTs seem to be limited to yeasts and plants (8). Interestingly, whereas type I and II enzymes catalyze MMA production in addition ...

In the family of protein arginine methyltransferases (PRMTs) that predominantly generate either asymmetric or symmetric dimethylarginine (SDMA), PRMT7 is unique in producing solely monomethylarginine (MMA) products. The type of methylation on histones and other proteins dictates changes in gene expression, and numerous studies have linked altered profiles of methyl marks with disease phenotypes. Given the importance of specific inhibitor development, it is crucial to understand the mechanisms by which PRMT product specificity is conferred. We have focused our attention on active-site residues of PRMT7 from the protozoan Trypanosoma brucei. We have designed 26 single and double mutations in the active site, including residues in the Glu-Xaa 8 -Glu (double E) loop and the Met-Gln-Trp sequence of the canonical Thr-His-Trp (THW) loop known to interact with the methyl-accepting substrate arginine. Analysis of the reaction products by high resolution cation exchange chromatography combined with the knowledge of PRMT crystal structures suggests a model where the size of two distinct subregions in the active site determines PRMT7 product specificity.