Ralf Ficner

Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'-phosphopantetheinyl transferase superfamily.

The Bacillus subtilis Sfp protein activates the peptidyl carrier protein (PCP) domains of surfactin synthetase by transferring the 4′‐phosphopantetheinyl moiety of coenzyme A (CoA) to a serine residue conserved in all PCPs. Its wide PCP substrate spectrum renders Sfp a biotechnologically valuable enzyme for use in combinatorial non‐ribosomal peptide synthesis. The structure of the Sfp–CoA complex determined at 1.8 Å resolution reveals a novel α/β‐fold exhibiting an unexpected intramolecular 2‐fold pseudosymmetry. This suggests a similar fold and dimerization mode for the homodimeric phosphopantetheinyl transferases such as acyl carrier protein synthase. The active site of Sfp accommodates a magnesium ion, which is complexed by the CoA pyrophosphate, the side chains of three acidic amino acids and one water molecule. CoA is bound in a fashion that differs in many aspects from all known CoA–protein complex structures. The structure reveals regions likely to be involved in the interaction with the PCP substrate.

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6·U5] tri‐snRNP that binds directly to the 5′ stem–loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP‐associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine‐rich loop flanked by the stem of the 5′ stem–loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5′ stem–loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre‐mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5′ stem–loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac·U5] tri‐snRNP of the minor U12‐type spliceosome.

NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1

Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP−CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.

Crystal Structure of the Nuclear Export Receptor CRM1 in Complex with Snurportin1 and RanGTP

Nuclear Import/Export Receptor Nuclear transport receptors constantly shuttle cargo between the nucleus and the cytoplasm through nuclear pore complexes. In the nucleus, RanGTP promotes the dissociation of cargo from importins, which import cargo into the nucleus (where RanGTP is guanosine 5′ triphosphate–bound Ran). Conversely, nuclear RanGTP promotes cargo-binding to exportins, which export cargo from the nucleus. Cargo is released from exportins in the cytoplasm upon hydrolysis of RanGTP. Cytoplasmically assembled RNA splicing components enter the nucleus together with an import adapter snurportin 1 (SPN1), but how then does the import adapter release its cargo and exit the nucleus to collect further cargo? The nuclear exportin CRM1 exports a broad range of substrates—including SPN1, ribosomes, and many regulatory proteins. Monecke et al. (p. 1087) describe the crystal structure of CRM1 bound to SPN1 and RanGTP. The structure shows that SPN1 cannot simultaneously bind its import cargo and the exportin CRM1, ensuring that only cargo-free SPN1 is returned to the cytoplasm. There are no direct contacts between Ran and SPN1 in the ternary complex, suggesting that RanGTP promotes cargo-binding through long-range conformational changes in CRM1. The structure of an exportin complex shows how nuclear transport complexes differentially recognize cargo. CRM1 mediates nuclear export of numerous unrelated cargoes, which may carry a short leucine-rich nuclear export signal or export signatures that include folded domains. How CRM1 recognizes such a variety of cargoes has been unknown up to this point. Here we present the crystal structure of the SPN1⋅CRM1⋅RanGTP export complex at 2.5 angstrom resolution (where SPN1 is snurportin1 and RanGTP is guanosine 5′ triphosphate–bound Ran). SPN1 is a nuclear import adapter for cytoplasmically assembled, m3G-capped spliceosomal U snRNPs (small nuclear ribonucleoproteins). The structure shows how CRM1 can specifically return the cargo-free form of SPN1 to the cytoplasm. The extensive contact area includes five hydrophobic residues at the SPN1 amino terminus that dock into a hydrophobic cleft of CRM1, as well as numerous hydrophilic contacts of CRM1 to m3G cap-binding domain and carboxyl-terminal residues of SPN1. The structure suggests that RanGTP promotes cargo-binding to CRM1 solely through long-range conformational changes in the exportin.

Crystal structure of the polysialic acid-degrading endosialidase of bacteriophage K1F.

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple β-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.

Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.

The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.

Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme.

Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.

Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2·2 Å resolution

The light-harvesting pigment-protein complex B-phycoerythrin from the red alga Porphyridium sordidum has been isolated and crystallized. B-Phycoerythrin consists of three different subunits forming an (alpha beta)6 gamma aggregate. The three-dimensional structure of the (alpha beta)6 hexamer was solved by Patterson search techniques using the molecular model of C-phycocyanin from Fremyella diplosiphon. The asymmetric unit of the crystal cell (space group P3, with a = b = 111.2 A, c = 59.9 A, alpha = beta = 90 degrees, gamma = 120 degrees) contains two (alpha beta) monomers related by a local dyad. Three asymmetric units are arranged around the crystallographic 3-fold axis building an (alpha beta)6 hexamer, as in C-phycocyanin. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional R-factor of the final model was 18.9% with data to 2.2 A resolution. The molecular structures of the alpha and beta-subunits resemble those of C-phycocyanin. Major changes in comparison to phycocyanin are caused by deletion or insertion of segments involved in protein-chromophore interactions. The singly linked phycoerythrobilin chromophores alpha-84, alpha-140a, beta-84 and beta-155 are each covalently bound to a cysteine by ring A. The doubly linked chromophore beta-50/beta-61 is attached at cysteine beta-50 through ring A and at cysteine beta-61 through ring D. B-Phycoerythrin contains additionally a 30 kDa gamma-subunit, which is presumably located in the central cavity of the hexamer. It is disordered, as a consequence of crystal and local symmetry averaging.

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA‐guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine‐34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine‐34 with the queuine (.)precursor 7‐aminomethyl‐7‐deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R‐factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8‐barrel with a tightly attached C‐terminal zinc‐containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha‐helix, located close to the C‐terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA‐enzyme intermediate. This structure is the first example of a (beta/alpha)‐barrel protein interacting specifically with a nucleic acid.

The iron-sulphur protein RNase L inhibitor functions in translation termination

The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.