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Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on the Escherichia coli structure. Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of the Y. pestis TesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Three complete genomes of beak and feather disease virus (BFDV) were recovered from wild twenty-eight parrots (Polytelis anthopeplus monarchoides). The genomes consisted of 1,996 bp with 1,934 identical sites and a typically content stem-loop structure between ORF1 and ORF2. This is the first report of BFDV infection as well as the complete genome sequences for this host species globally., (Copyright © 2014 Sarker et al.)

Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe(154)-Thr(178)), defined here as RegLoop1, and a second region involving the C terminus of thioesterase domain 2 (Gln(304)-Gly(326)), designated RegLoop2. Mutagenesis confirmed that Arg(312) and Arg(313) are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

The complete genome sequence of beak and feather disease virus (BFDV) from a wild Australian Mallee ringneck parrot (Barnardius zonarius barnardi) was characterized. The genome consists of 1,995 nucleotides and encodes two major proteins in opposing directions. This is the first evidence of BFDV infectivity and the first complete genome sequence for this novel host., (Copyright © 2014 Das et al.)

Phylogenetic analyses of the highly genetically diverse but antigenically conserved, single-stranded circular, DNA genome of the avian circovirus, beak and feather disease virus (BFDV) from cockatoo species throughout Australia demonstrated a high mutation rate for BFDV (orders of magnitude fall in the range of 10(-4) substitutions/site/year) along with strong support for recombination indicating active cross-species transmission in various subpopulations. Multiple variants of BFDV were demonstrated with at least 30 genotypic variants identified within nine individual birds, with one containing up to 7 variants. Single genetic variants were detected in feathers from 2 birds but splenic tissue provided further variants. The rich BFDV genetic diversity points to Australasia as the most likely geographical origin of this virus and supports flexible host switching. We propose this as evidence of Order-wide host generalism in the Psittaciformes characterised by high mutability that is buffered by frequent recombination and slow replication strategy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Miltefosine (MI) is a novel, potential antifungal agent with activity against some yeast and filamentous fungal pathogens. We previously demonstrated in the model yeast, Saccharomyces cerevisiae, that MI causes disruption of mitochondrial membrane potential and apoptosis-like cell death via interaction with the Cox9p sub-unit of cytochrome c oxidase (COX). To identify additional mechanisms of antifungal action, MI resistance was induced in S. cerevisiae by exposure to the mutagen, ethyl methanesulfonate, and gene mutation(s) responsible for resistance were investigated. An MI-resistant haploid strain (H-C101) was created. Resistance was retained in the diploid strain (D-C101) following mating, confirming dominant inheritance. Phenotypic assessment of individual D-C101 tetrads revealed that only one mutant gene contributed to the MI-resistance phenotype. To identify this gene, the genome of H-C101 was sequenced and 17 mutated genes, including metacaspase-encoding MCA1, were identified. The MCA1 mutation resulted in substitution of asparagine (N) with aspartic acid (D) at position 164 (MCA1(N164D)). MI resistance was found to be primarily due to MCA1(N164D), as single-copy episomal expression of MCA1(N164D), but not two other mutated genes (FAS1(T1417I) and BCK2(T104A)), resulted in MI resistance in the wild-type strain. Furthermore, an MCA1 deletion mutant (mca1Δ) was MI-resistant. MI treatment led to accumulation of reactive oxygen species (ROS) in MI-resistant (MCA1(N164D)-expressing and mca1Δ) strains and MI-susceptible (MCA1-expressing) strains, but failed to activate Mca1 in the MI-resistant strains, demonstrating that ROS accumulation does not contribute to the fungicidal effect of MI. In conclusion, functional disruption of Mca1, leads to MI resistance and inability to mediate MI-induced apoptotic effects. Mca1-mediated apoptosis is therefore a major mechanism of MI-induced antifungal action., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-related N-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT from S. aureus (SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space group P43212, with unit-cell parameters a = b = 84.86, c = 49.06 Å, α = β = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.

Staphylococcus aureus is the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) from S. aureus subsp. aureus Mu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization of SaPaaI thioesterase from S. aureus are reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mM sodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space group P12(1)1, with unit-cell parameters a = 44.05, b = 89.05, c = 60.74 Å, β = 100.5°. Initial structure determination and refinement gave an R factor and R(free) of 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.

The whole-genome sequence of beak and feather disease virus (BFDV) from a wild Australian regent parrot (Polytelis anthopeplus monarchoides) was characterized. The genome consists of 1,993 bp and has a typical stem-loop structure between open reading frame 1 (ORF1) and ORF2. This is the first evidence of BFDV infection as well as the complete genome sequence for this host species, globally.

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10(-3) subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.

The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported. Recombinant FabG was expressed, purified to homogeneity and crystallized via the hanging-drop vapour-diffusion technique. Diffraction data were collected at the Australian Synchrotron to 2.30 Å resolution. The crystal displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 68.22, b = 98.68, c = 169.84 Å, and four ypFabG molecules in the asymmetric unit.

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Å(2) of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters (17)RAKKMSK(23) and (3)KR(4), respectively. One notable feature of the major site is the insertion of Prp20NLS Ala(18) between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.

Two complete genomes of beak and feather disease virus (BFDV) were characterized from Lathamus discolor, the Australian swift parrot. This is the first report of BFDV complete genome sequences in this host. The completed BFDV genomes consist of 1,984 nucleotides encoding two open reading frames with 99.7% pairwise nucleotide identity.

The complete genome sequence of a beak and feather disease virus (BFDV) encoding two major open reading frames (ORFs) was characterized in a wild Moluccan red lory (Eos bornea). This is the first report of a BFDV genome from Indonesia and the first reported BFDV infection for this host species.

Neisseria meningitidis is the causative microorganism of many human diseases, including bacterial meningitis; together with Streptococcus pneumoniae, it accounts for approximately 80% of bacterial meningitis infections. The emergence of antibiotic-resistant strains of N. meningitidis has created a strong urgency for the development of new therapeutics, and the high-resolution structural elucidation of enzymes involved in cell metabolism represents a platform for drug development. Acetyl-CoA hydrolase is involved in multiple functions in the bacterial cell, including membrane synthesis, fatty-acid and lipid metabolism, gene regulation and signal transduction. Here, the first recombinant protein expression, purification and crystallization of a hexameric acetyl-CoA hydrolase from N. meningitidis are reported. This protein was crystallized using the hanging-drop vapour-diffusion technique at pH 8.5 and 290 K using ammonium phosphate as a precipitant. Optimized crystals diffracted to 2.0 Å resolution at the Australian Synchrotron and belonged to space group P2(1)3 (unit-cell parameters a = b = c = 152.2 Å), with four molecules in the asymmetric unit.

Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Expression of recombinant beak and feather disease virus (BFDV) capsid-associated protein (Cap) has relied on inefficient techniques that typically produce low yields or use specialized expression systems, which greatly increase the cost and expertise required for mass production. An Escherichia coli system was used to express recombinant BFDV Cap derived from two isolates of BFDV, from a Long-billed Corella (Cacatua tenuirostris) and an Orange-bellied parrot (OBP; Neophema chrysogaster). Purification by affinity and size exclusion chromatography was optimized through an iterative process involving screening and modification of buffer constituents and pH. A buffer containing glycerol, β-mercaptoethanol, Triton X-100, and a high concentration of NaCl at pH 8 was used to increase solubility of the protein. The final concentration of the corella-isolated BFDV protein was fifteen- to twenty-fold greater than that produced in previous publications using E. coli expression systems. Immunoassays were used to confirm the specific antigenicity of recombinant Cap, verifying its validity for use in continued experimentation as a potential vaccine, a reagent in diagnostic assays, and as a concentrated sample for biological discoveries., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Yersinia pestis is a highly virulent human pathogen and is the causative agent of bubonic plague. Spread through the bite of infected fleas, plague epidemics have marked important events in history, including the Justinian plague (6th century), the Black Death (14th century) which decimated nearly one quarter of the European population, and more recently the Orientalis plague (1894). To date, deaths are still being reported and, without treatment, the disease kills most people within 4 days. One of the thioesterases from Y. pestis, TesB, is a broad-range acyl-CoA thioesterase and is highly conserved within prokaryotes and throughout evolution, sharing sequence similarity with the HIV Nef binding protein ACOT8. Here the expression, purification, crystallization and diffraction of TesB are reported. TesB has been recombinantly expressed and crystallized using the vapour-diffusion hanging-drop technique at pH 7.0 and 290 K. After optimization, crystals diffracted to 2.0 Å resolution at the Australian Synchrotron and belong to the space group P12(1)1 (a = 73.55, b = 170.82, c = 101.98 Å), with eight molecules likely to be present in the asymmetric unit.

Classical nuclear localization signals (cNLSs), comprising one (monopartite cNLSs) or two clusters of basic residues connected by a 10-12 residue linker (bipartite cNLSs), are recognized by the nuclear import factor importin-α. The cNLSs bind along a concave groove on importin-α; however, specificity determinants of cNLSs remain poorly understood. We present a structural and interaction analysis study of importin-α binding to both designed and naturally occurring high-affinity cNLS-like sequences; the peptide inhibitors Bimax1 and Bimax2, and cNLS peptides of cap-binding protein 80. Our data suggest that cNLSs and cNLS-like sequences can achieve high affinity through maximizing interactions at the importin-α minor site, and by taking advantage of multiple linker region interactions. Our study defines an extended set of binding cavities on the importin-α surface, and also expands on recent observations that longer linker sequences are allowed, and that long-range electrostatic complementarity can contribute to cNLS-binding affinity. Altogether, our study explains the molecular and structural basis of the results of a number of recent studies, including systematic mutagenesis and peptide library approaches, and provides an improved level of understanding on the specificity determinants of a cNLS. Our results have implications for identifying cNLSs in novel proteins., (© 2011 John Wiley & Sons A/S.)

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.

Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-β/karyopherin-β1; and (ii) the karyopherin-β2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-β2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-α•cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import., (2010 Elsevier B.V. All rights reserved.)

Acyl-coenzyme A thioesterases (Acots) play important cellular roles in mammalian fatty acid metabolism through modulation of cellular concentrations of activated fatty acyl-CoAs. Acots catalyse the hydrolysis of the thioester bond present within acyl-CoA ester molecules to yield coenzyme A (CoASH) and the corresponding non-esterified fatty acid. Acyl-CoA thioesterases are expressed ubiquitously in both prokaryotes and eukaryotes and, in higher order organisms, the enzymes are expressed and localised in a tissue-dependent manner within the cytosol, mitochondria, peroxisomes and endoplasmic reticulum. Recent studies have led to advances in the functional and structural characterization of many mammalian Acot family members. These include the structure determination of both type-I and type-II Acot family members, structural elucidation of the START domain of ACOT11, identification of roles in arachidonic acid and inflammatory prostaglandin production by Acot7, and inclusion of a 13th Acot family member. Here, we review and analyse the current literature on mammalian Acots with respect to their characterization and summarize the current knowledge on the structure, function and regulation of this enzyme family., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The structure of solenoid proteins facilitates a higher degree of flexibility than most folded proteins. In importin-β, a nuclear import factor built from 19 tandem HEAT repeats, flexibility plays a crucial role in allowing interactions with a range of different partners. We present a comprehensive analysis of importin-β flexibility based on a number of different approaches. We determined the crystal structure of unliganded Saccharomyces cerevisiae importin-β (Kap95) to allow a quantitative comparison with importin-β bound to different partners. Complementary mutagenesis, small angle X-ray scattering and molecular dynamics studies suggest that the protein samples several conformations in solution. The analyses suggest the flexibility of the solenoid is generated by cumulative small movements along its length. Importin-β illustrates how solenoid proteins can orchestrate protein interactions in many cellular pathways., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located 'beta-NLS' that mediates nuclear transport through Impbeta1 (importin beta1) and the N-terminally located 'CaM-NLS' which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)-SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors.

Background: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate., Methodology/principal Findings: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised., Conclusions/significance: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

Ran-GTP interacts strongly with importin-beta, and this interaction promotes the release of the importin-alpha-nuclear localization signal cargo from importin-beta. Ran-GDP also interacts with importin-beta, but this interaction is 4 orders of magnitude weaker than the Ran-GTP.importin-beta interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-beta promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-beta complex stabilizes the complex, which cannot be dissociated by importin-alpha. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-beta has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-beta. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-beta. Importin-alpha inhibits the GDP exchange reaction by sequestering importin-beta, whereas RanBP1 restores the GDP nucleotide exchange by importin-beta by forming a tetrameric complex with importin-beta, Ran, and importin-alpha. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.

The transport of macromolecules across the nuclear envelope is an essential eukaryotic process that enables proteins such as transcription factors, polymerases and histones to gain access to the genetic material contained within the nucleus. Importin-beta plays a central role in the nucleocytoplasmic transport process, mediating nuclear import through a range of interactions with cytoplasmic, nuclear and nuclear pore proteins such as importin-alpha, Ran, nucleoporins and various cargo molecules. The unliganded form of the full-length yeast importin-beta has been expressed and crystallized. The crystals were obtained by vapour diffusion at pH 6.5 and 290 K. The crystals belonged to space group P2(1) (unit-cell parameters a = 58.17, b = 127.25, c = 68.52 A, beta = 102.23). One molecule is expected in the asymmetric unit. The crystals diffracted to 2.4 A resolution using a laboratory X-ray source and were suitable for crystal structure determination.

Crystallography is commonly used for studying the structures of protein-protein complexes. However, a crystal structure does not define a unique protein-protein interface, and distinguishing a 'biological interface' from 'crystal contacts' is often not straightforward. A number of computational approaches exist for distinguishing them, but their error rate is high, emphasizing the need to obtain further data on the biological interface using complementary structural and functional approaches. In addition to reviewing the computational and experimental approaches for addressing this problem, we highlight two relevant examples. The first example from our laboratory involves the structure of acyl-CoA thioesterase 7, where each domain of this two-domain protein was crystallized separately, but both yielded a non-functional assembly. The structure of the full-length protein was uncovered using a combination of complementary approaches including chemical cross-linking, analytical ultracentrifugation and mutagenesis. The second example involves the platelet glycoprotein Ibalpha-thrombin complex. Two groups reported the crystal structures of this complex, but all the interacting interfaces differed between the two structures. Our computational analysis did not fully resolve the reasons for the discrepancies, but provided interesting insights into the system. This review highlights the need to complement crystallographic studies with complementary experimental and computational approaches.

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5'-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5'-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-beta. Here, we show that RanGDP and importin-beta form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-beta binding partners such as importin-alpha. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation., (2007 Wiley-Liss, Inc.)

Cross-linking in combination with mass spectrometry can be used as a tool for structural modeling of protein complexes and multidomain proteins. Although cross-links represent only weak structural constraints, the combination of a limited set of experimental cross-links with molecular docking/modeling is often sufficient to determine the structure of a protein complex or multidomain protein at low resolution.

This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.

Increasing the success in obtaining structures and maximizing the value of the structures determined are the two major goals of target selection in structural proteomics. This chapter presents an efficient and flexible target selection procedure supplemented with a Web-based resource that is suitable for small- to large-scale structural genomics projects that use crystallography as the major means of structure determination. Based on three criteria, biological significance, structural novelty, and "crystallizability," the approach first removes (filters) targets that do not meet minimal criteria and then ranks the remaining targets based on their "crystallizability" estimates. This novel procedure was designed to maximize selection efficiency, and its prevailing criteria categories make it suitable for a broad range of structural proteomics projects.

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

Metal-binding sites are ubiquitous in proteins and can be readily utilized for phasing. It is shown that a protein crystal structure can be solved using single-wavelength anomalous diffraction based on the anomalous signal of a cobalt ion measured on a conventional monochromatic X-ray source. The unique absorption edge of cobalt (1.61 A) is compatible with the Cu K alpha wavelength (1.54 A) commonly available in macromolecular crystallography laboratories. This approach was applied to the determination of the structure of Melampsora lini avirulence protein AvrL567-A, a protein with a novel fold from the fungal pathogen flax rust that induces plant disease resistance in flax plants. This approach using cobalt ions may be applicable to all cobalt-binding proteins and may be advantageous when synchrotron radiation is not readily available.

The sex-determining factor SRY plays an important role in male sexual development, diverting primordial gonads from the ovarian pathway toward male differentiation to form testes. SRY is a DNA-binding protein and gains access to the nucleus through two independently acting nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain. We have reconstituted the nuclear import of SRY using an in vitro nuclear transport assay, showing that nuclear import of SRY can occur in the absence of additional exogenous cytosolic factors, with a significant reduction in nuclear transport in the presence of antibodies to the nuclear transport protein importin (Imp) beta1 but not Impalpha. We have also shown using in vitro binding assays that the C-terminal NLS of SRY binds directly to Impbeta1. Finally, we have shown that SRY can target green fluorescent protein to the nucleus in a mammalian transfected cell line; importantly, mutations known to result in sex reversal that map to either NLS impair nuclear accumulation implying that SRY nuclear import is critical to its function.

The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 A, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 A resolution using the laboratory X-ray source and are suitable for crystal structure determination.

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 A r.m.s. deviation of Calpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.

The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding/bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMPbeta. The SRY-R133W mutant shows reduced IMPbeta binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMPbeta binding, whereas SRY-R75N and SRY-R76P show normal IMPbeta binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

Telomere repeat factor 1 (TRF1) regulates the steady-state length of chromosomes, whereby its overexpression results in telomere shortening while dominant negative TRF1 mutations can lead to telomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleus at mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism by which TRF1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, in turn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism of TRF by expressing and purifying a recombinant TRF1-GFP (green fluorescent protein) fusion protein that is functional in terms of being able to bind telomeric DNA specifically as shown using a novel, quantitative double-label gel mobility shift assay. We quantitate the ability of TRF1-GFP to accumulate in the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathway of TRF1 is mediated by importin (Imp) beta1 and Ran. Imp beta is shown to bind directly to TRF1 with nanomolar affinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experiments also demonstrate that Imp beta residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerized to Imp beta, Imp alpha was found to inhibit Imp beta-mediated nuclear accumulation, although not affecting Imp beta binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1; that its nuclear import is mediated directly by Imp beta but inhibited by Imp alpha may represent a novel regulatory mechanism, with potential relevance to oncogenesis.

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.