tase genes have revealed a conserved and ordered modular 4 Corresponding author organization. Each module encodes a functional building unit containing~1000 amino acids, which specifically The non-ribosomal synthesis of the cyclic peptide recognizes a single amino acid. Within such a protein antibiotic gramicidin S is accomplished by two large template-directed peptide biosynthesis, the occurrence and multifunctional enzymes, the peptide synthetases 1 and specific order of the modules in the genomic DNA dictate 2. The enzyme complex contains five conserved subunits the number and sequence of the amino acids to be of~60 kDa which carry out ATP-dependent activation incorporated into the resulting oligopeptide. The modular of specific amino acids and share extensive regions of arrangement of peptide synthetases closely parallels the sequence similarity with adenylating enzymes such multienzyme complexes responsible for the biogenesis of as firefly luciferases and acyl-CoA ligases. We have fatty acids and of the polyketide family of natural products. determined the crystal structure of the N-terminal Furthermore, peptide synthetases, fatty-acid synthetases adenylation subunit in a complex with AMP and and polyketide synthetases all use enzyme-bound phospho-L-phenylalanine to 1.9 Å resolution. The 556 amino pantetheine cofactors as acyl carriers, in a thiotemplate acid residue fragment is folded into two domains with mechanism first proposed by Lipmann more than 20 the active site situated at their interface. Each domain years ago (Lipmann, 1971) and revised recently (Stein of the enzyme has a similar topology to the correspond et al., 1996). ing domain of unliganded firefly luciferase, but aIn particular, the synthesis of the cyclic antibiotic remarkable relative domain rotation of 94°occurs.gramicidin S has been studied in detail. Gramicidin S is This conformation places the absolutely conserved produced by the Gram-positive bacterium Bacillus brevis Lys517 in a position to form electrostatic interactions and consists of two identical pentapeptides joined head with both ligands. The AMP is bound with the phosto tail. It is synthesized by the multienzyme complex phate moiety interacting with Lys517 and the hydroxyl gramicidin S synthetase, which is encoded by the 19 kb groups of the ribose forming hydrogen bonds with grs operon that includes the genes grsA, grsB and grsT. Asp413. The phenylalanine substrate binds in a hydro-The grsT gene, which is located at the 5Ј-end of the grs phobic pocket with the carboxylate group interacting operon, encodes a 29 kDa protein homologous to fattywith Lys517 and the α-amino group with Asp235. The acid thioesterases. The grsA gene product, gramicidin S structure reveals the role of the invariant residues synthetase 1 (GrsA) is a protein composed of 1098 amino within the superfamily of adenylate-forming enzymes acids (Hori et al., 1989; Krätzschmar et al., 1989). and indicates a conserved mechanism of nucleotide GrsA activates L-phenylalanine to the corresponding acylbinding and ...

Selective nuclear import is mediated by nuclear localization signals (NLSs) and cognate transport factors known as karyopherins or importins. Karyopherin alpha recognizes "classical" monopartite and bipartite NLSs. We report the crystal structure of a 50 kDa fragment of the 60 kDa yeast karyopherin alpha, in the absence and presence of a monopartite NLS peptide at 2.2 A and 2.8 A resolution, respectively. The structure shows a tandem array of ten armadillo repeats, organized in a right-handed superhelix of helices. Binding of the NLS peptide occurs at two sites within a helical surface groove that is lined by conserved residues. The structure reveals the determinants of NLS specificity and suggests a model for the recognition of bipartite NLSs.

Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. We have uncovered the molecular mechanism of Aurora-A activation by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that we identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation.

Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

In eukaryotic cells, segregation of DNA replication and RNA biogenesis in the nucleus and protein synthesis in the cytoplasm poses the requirement of transporting thousands of macromolecules between the two cellular compartments. Transport between nucleus and cytoplasm is mediated by soluble receptors that recognize specific cargoes and carry them through the nuclear pore complex (NPC), the sole gateway between the two compartments at interphase. Nucleocytoplasmic transport is specific not only in terms of cargo recognition, but also in terms of directionality, with nuclear proteins imported into the nucleus and RNAs exported from it. How is directionality achieved? How can the receptors be both specific and versatile in recognizing a multitude of cargoes? And how can their interaction with NPCs allow fast translocation? We describe the molecular mechanisms underlying nucleocytoplasmic transport as they have been revealed by structural studies of the receptors and regulators in different steps of transport cycles.