Showing papers in "Nature Structural & Molecular Biology in 2003"

Announcing the worldwide Protein Data Bank.

Abstract: mentation will be kept publicly available and the distribution sites will mirror the PDB archive using identical contents and subdirectory structure. However, each member of the wwPDB will be able to develop its own web site, with a unique view of the primary data, providing a variety of tools and resources for the global community. An Advisory Board consisting of appointees from the wwPDB, the International Union of Crystallography and the International Council on Magnetic Resonance in Biological Systems will provide guidance through annual meetings with the wwPDB consortium. This board is responsible for reviewing and determining policy as well as providing a forum for resolving issues related to the wwPDB. Specific details about the Advisory Board can be found in the wwPDB charter, available on the wwPDB web site. The RCSB is the ‘archive keeper’ of wwPDB. It has sole write access to the PDB archive and control over directory structure and contents, as well as responsibility for distributing new PDB identifiers to all deposition sites. The PDB archive is a collection of flat files in the legacy PDB file format 3 and in the mmCIF 4 format that follows the PDB exchange dictionary (http://deposit.pdb.org/ mmcif/). This dictionary describes the syntax and semantics of PDB data that are processed and exchanged during the process of data annotation. It was designed to provide consistency in data produced in structure laboratories, processed by the wwPDB members and used in bioinformatics applications. The PDB archive does not include the websites, browsers, software and database query engines developed by researchers worldwide. The members of the wwPDB will jointly agree to any modifications or extensions to the PDB exchange dictionary. As data technology progresses, other data formats (such as XML) and delivery methods may be included in the official PDB archive if all the wwPDB members concur on the alteration. Any new formats will follow the naming and description conventions of the PDB exchange dictionary. In addition, the legacy PDB format would not be modified unless there is a compelling reason for a change. Should such a situation occur, all three wwPDB members would have to agree on the changes and give the structural biology community 90 days advance notice. The creation of the wwPDB formalizes the international character of the PDB and ensures that the archive remains single and uniform. It provides a mechanism to ensure consistent data for software developers and users worldwide. We hope that this will encourage individual creativity in developing tools for presenting structural data, which could benefit the scientific research community in general.

Evolutionarily conserved networks of residues mediate allosteric communication in proteins

Abstract: A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originating at one site in a protein propagate reliably to affect distant functional sites. The general principles of protein structure that underlie this process remain unknown. Here, we describe a sequence-based statistical method for quantitatively mapping the global network of amino acid interactions in a protein. Application of this method for three structurally and functionally distinct protein families (G protein–coupled receptors, the chymotrypsin class of serine proteases and hemoglobins) reveals a surprisingly simple architecture for amino acid interactions in each protein family: a small subset of residues forms physically connected networks that link distant functional sites in the tertiary structure. Although small in number, residues comprising the network show excellent correlation with the large body of mechanistic data available for each family. The data suggest that evolutionarily conserved sparse networks of amino acid interactions represent structural motifs for allosteric communication in proteins.

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

Abstract: RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.

Phylogenomics of the nucleosome.

Abstract: Histones are best known as the architectural proteins that package the DNA of eukaryotic organisms, forming octameric nucleosome cores that the double helix wraps tightly around Although histones have traditionally been viewed as slowly evolving scaffold proteins that lack diversification beyond their abundant tail modifications, recent studies have revealed that variant histones have evolved for diverse functions H2A and H3 variants have diversified to assume roles in epigenetic silencing, gene expression and centromere function Such diversification of histone variants and 'deviants' contradicts the perception of histones as monotonous members of multigene families that indiscriminately package and compact the genome How these diverse functions have evolved from ancestral forms can be addressed by applying phylogenetic tools to increasingly abundant sequence data

The mechanical stability of ubiquitin is linkage dependent.

Abstract: Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C–linked and Lys48-C–linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Abstract: Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The � -helical ‘stem’ regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The ‘anchor’ regions of E and the M proteins each form antiparallel E-E and M-M transmembrane � -helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.

An mRNA structure that controls gene expression by binding S-adenosylmethionine.

Abstract: Riboswitches are metabolite-binding RNA structures that serve as genetic control elements for certain messenger RNAs. These RNA switches have been identified in all three kingdoms of life and are typically responsible for the control of genes whose protein products are involved in the biosynthesis, transport or utilization of the target metabolite. Herein, we report that a highly conserved RNA domain found in bacteria serves as a riboswitch that responds to the coenzyme S-adenosylmethionine (SAM) with remarkably high affinity and specificity. SAM riboswitches undergo structural reorganization upon introduction of SAM, and these allosteric changes regulate the expression of 26 genes in Bacillus subtilis. This and related findings indicate that direct interaction between small metabolites and allosteric mRNAs is an important and widespread form of genetic regulation in bacteria.

Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

Abstract: The facultative anaerobe Escherichia coli is able to assemble specific respiratory chains by synthesis of appropriate dehydrogenases and reductases in response to the availability of specific substrates. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. We present here the crystal structure of NarGHI at a resolution of 1.9 A. The NarGHI structure identifies the number, coordination scheme and environment of the redox-active prosthetic groups, a unique coordination of the molybdenum atom, the first structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor in the catalytic mechanism and a novel fold of the membrane anchor subunit. Our findings provide fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop.

Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity.

Abstract: The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.

Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains

Abstract: The ATPase p97/VCP affects multiple events within the cell. These events include the alteration of both nuclear and mitotic Golgi membranes, the dislocation of ubiquitylated proteins from the endoplasmic reticulum and regulation of the NF-κb pathway. Here we present the crystal structure of full-length Mus musculus p97/VCP in complex with a mixture of ADP and ADP–AlFx at a resolution of 4.7 A. This is the first complete hexameric structure of a protein containing tandem AAA (ATPases associated with a variety of cellular activities) domains. Comparison of the crystal structure and cryo-electron microscopy (EM) reconstructions reveals large conformational changes in the helical subdomains during the hydrolysis cycle. Structural and functional data imply a communication mechanism between the AAA domains. A Zn2+ occludes the central pore of the hexamer, suggesting that substrate does not thread through the pore of the molecule.

Ni-Zn-[Fe4-S4] and Ni-Ni-[Fe4-S4] clusters in closed and open subunits of acetyl-CoA synthase/carbon monoxide dehydrogenase.

Abstract: The crystal structure of the tetrameric α2β2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two α subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the α subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog.

Abstract: Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.

Insights into antifolate resistance from malarial DHFR-TS structures.

Abstract: Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.

Pulling geometry defines the mechanical resistance of a β-sheet protein

Abstract: Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-� protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

Automated design of specificity in molecular recognition.

Abstract: Specific protein–protein interactions are crucial in signaling networks and for the assembly of multi-protein complexes, and represent a challenging goal for protein design. Optimizing interaction specificity requires both positive design, the stabilization of a desired interaction, and negative design, the destabilization of undesired interactions. Currently, no automated protein-design algorithms use explicit negative design to guide a sequence search. We describe a multi-state framework for engineering specificity that selects sequences maximizing the transfer free energy of a protein from a target conformation to a set of undesired competitor conformations. To test the multi-state framework, we engineered coiled-coil interfaces that direct the formation of either homodimers or heterodimers. The algorithm identified three specificity motifs that have not been observed in naturally occurring coiled coils. In all cases, experimental results confirm the predicted specificities.

The CTD code.

Abstract: How does the C-terminal domain (CTD) of RNA polymerase II interact specifically with multiple targets? A recent paper describing the structure of this domain with a mRNA capping enzyme guanylyltransferase suggests that the CTD is a contortionist that, upon post-translational modification, adopts different configurations specifically recognized by its partners.

The crystal structure of the proprotein processing proteinase furin explains its stringent specificity.

Abstract: In eukaryotes, many essential secreted proteins and peptide hormones are excised from larger precursors by members of a class of calcium-dependent endoproteinases, the prohormone-proprotein convertases (PCs). Furin, the best-characterized member of the mammalian PC family, has essential functions in embryogenesis and homeostasis but is also implicated in various pathologies such as tumor metastasis, neurodegeneration and various bacterial and viral diseases caused by such pathogens as anthrax and pathogenic Ebola virus strains. Furin cleaves protein precursors with narrow specificity following basic Arg-Xaa-Lys/Arg-Arg-like motifs. The 2.6 A crystal structure of the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)−inhibited mouse furin ectodomain, the first PC structure, reveals an eight-stranded jelly-roll P domain associated with the catalytic domain. Contoured surface loops shape the active site by cleft, thus explaining furin's stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furin's preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.

Structural dynamics of individual Holliday junctions.

Abstract: The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.

Incorporation of Aminoacyl-tRNA into the Ribosome as seen by Cryo-electron Microscopy

Abstract: Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.

Correction of disease-associated exon skipping by synthetic exon-specific activators

Abstract: Differential exon use is a hallmark of alternative splicing, a prevalent mechanism for generating protein isoform diversity. Many disease-associated mutations also affect pre-mRNA splicing, usually causing inappropriate exon skipping. SR proteins are essential splicing factors that recognize exonic splicing enhancers and drive exon inclusion. To emulate this function of SR proteins, we designed small chimeric effectors comprising a minimal synthetic RS domain covalently linked to an antisense moiety that targets an exon by Watson-Crick base pairing. Here we show that such synthetic effectors can mimic the functions of SR proteins and specifically restore wild type splicing when directed to defective BRCA1 or SMN2 pre-mRNA transcripts. This general approach can be used as a tool to investigate splicing mechanisms and modulate alternative splicing of specific genes, and as a therapeutic strategy to correct splicing defects responsible for numerous diseases.

Translocation of botulinum neurotoxin light chain protease through the heavy chain channel.

Abstract: Clostridial botulinum neurotoxins (BoNTs) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis An enigmatic step in the intoxication process is the mechanism by which the neurotoxin heavy chain (HC) forms the conduit for the translocation of the light chain (LC) protease across the endosomal membrane into the cytosol, its site of action Here we investigate the mechanism of LC translocation by using the combined detection of channel currents and substrate proteolysis, the two hallmark activities of BoNT Our data are consistent with the translocation of the LC through the HC channel and show that the LC protease activity is retrieved in the trans compartment after translocation We propose that the BoNT HC–LC complex embedded in the membrane is a transmembrane chaperone, a dynamic structural device that prevents aggregation and achieves translocation of the LC In this regard, the complex is similar to the protein conducting/translocating channels of the endoplasmic reticulum, mitochondria and chloroplasts

Amyloid-like Filaments and Water-filled Nanotubes Formed by SOD1 Mutant Proteins Linked to Familial ALS

Abstract: Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned beta-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and beta-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal beta-sheet-containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

The structure and function of MCM from archaeal M.Thermoautotrophicum

Abstract: Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.

Structural insights into the U-box, a domain associated with multi-ubiquitination

Abstract: The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING–E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

Abstract: The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B12). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (Kd ∼0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.

Structure of HCV IRES domain II determined by NMR.

Abstract: Complex RNA structures regulate many biological processes, but are often too large for structure determination by NMR methods. The 5′ untranslated region (5′ UTR) of the hepatitis C viral (HCV) RNA genome contains an internal ribosome entry site (IRES) that binds to 40S ribosomal subunits with high affinity and specificity to control translation. Domain II of the HCV IRES forms a 25-kDa folded subdomain that may alter ribosome conformation. We report here the structure of domain II as determined using an NMR approach that combines short- and long-range structural data. Domain II adopts a distorted L-shape structure, and its overall shape in the free form is markedly similar to its 40S subunit–bound form; this suggests how domain II may modulate 40S subunit conformation. The results show how NMR can be used for structural analysis of large biological RNAs.

Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.

Abstract: Many bacteria and about 40,000 plant species form primary carbohydrate reserves based on fructan; these polymers of beta-D-fructofuranose are thought to confer tolerance to drought and frost in plants. Microbial fructan, the beta(2,6)-linked levan, is synthesized directly from sucrose by levansucrase, which is able to catalyze both sucrose hydrolysis and levan polymerization. The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 A, shows a rare five-fold beta-propeller topology with a deep, negatively charged central pocket. Arg360, a residue essential for polymerase activity, lies in a solvent-exposed site adjacent to the central pocket. Mutagenesis data and the sucrose-bound structure of inactive levansucrase E342A, at a resolution of 2.1 A, strongly suggest that three conserved acidic side chains in the central pocket are critical for catalysis, and presumably function as nucleophile (Asp86) and general acid (Glu342), or stabilize the transition state (Asp247).

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration

Abstract: The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1–vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 A of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion

Abstract: The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS–tRNATyr–L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNATyr identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Light-induced electron transfer in a cryptochrome blue-light photoreceptor.

Abstract: Cryptochromes are flavoproteins implicated in multiple blue light-dependent signaling pathways regulating, for example, photomorphogenesis in plants or circadian clocks in animals. Using transient absorption spectroscopy, it is demonstrated that the primary light reactions in isolated Arabidopsis thaliana cryptochrome-1 involve intraprotein electron transfer from tryptophan and tyrosine residues to the excited flavin adenine dinucleotide cofactor.