Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 µL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide.

The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform.

Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster.

The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7Å resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a ß-sheet and three α-helices forming a cone-like shape. However, known binding sites for RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.

Purification, crystallization and preliminary X-ray diffraction of the G3BP1 NTF2-like domain.

The nuclear transport factor 2-like (NTF2-like) domain of human G3BP1 was subcloned, overexpressed in Escherichia coli and purified. Crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected to 3.6 Šresolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a=b=89.84, c=70.02 Å. The crystals contained one molecule per asymmetric unit, with an estimated solvent content of 56%. Initial phases were obtained by molecular replacement.

Structure of a fatty-acid-binding protein from Bacillus subtilis determined by sulfur-SAD phasing using in-house chromium radiation.

Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 A resolution using Cr Kalpha radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family.

Structure of the first PDZ domain of human PSD-93.

The crystal structure of the PDZ1 domain of human PSD-93 has been determined to 2.0 A resolution. The PDZ1 domain forms a crystallographic trimer that is also predicted to be stable in solution. The main contributions to the stabilization of the trimer seem to arise from interactions involving the PDZ1-PDZ2 linker region at the extreme C-terminus of PDZ1, implying that the oligomerization that is observed is not of biological significance in full-length PSD-93. Comparison of the structures of the binding cleft of PSD-93 PDZ1 with the previously reported structures of PSD-93 PDZ2 and PDZ3 as well as of the closely related human PSD-95 PDZ1 shows that they are very similar in terms of amino-acid composition. However, the cleft is significantly narrower in PSD-95. This could be part of the basis of peptide selectivity between PSD-93 PDZ1 and PSD-95 PDZ1.

Structure of a SARS coronavirus-derived peptide bound to the human major histocompatibility complex class I molecule HLA-B*1501.

The human leukocyte antigen (HLA) class I system comprises a highly polymorphic set of molecules that specifically bind and present peptides to cytotoxic T cells. HLA-B*1501 is a prototypical member of the HLA-B62 supertype and only two peptide-HLA-B*1501 structures have been determined. Here, the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making it available for interactions with a potential T-cell receptor.

Danish general practitioners' professional attention to children of parents with depression.

INTRODUCTION: Offspring of parents with depression has an increased risk of experiencing somatic and psychiatric diseases. Early child support can reduce this risk. This study aimed to describe general practitioners' (GPs) professional attention to children of depressed patients. METHODS: This was a survey study. We mailed ques-tion-naires to randomly selected Danish GPs. RESULTS: Among the 1,760 GPs invited, 890 (51%) partici-pated. Female GPs accounted for 45% of the respondents and 41% of the total GP population (p = 0.02). Respondents were younger than the mean GP population. A total of 94% of the GPs reported that giving attention to children of de-pressed parents was relevant, and 65% reported addressing the children's well-being during the consultation with the parent. A total of 39% of the GPs found that their knowledge about the significance of parental depression for the child was poor, and 41% were highly interested in learning more. Female GPs perceived that they had sufficient knowledge (66%) more frequently than male GPs (56%) (p < 0.001). GPs with sufficient perceived knowledge addressed the children's well-being more frequently than GPs with poor perceived knowledge (odds ratio = 5.8; 95% confidence interval: 4.14-8.07). CONCLUSIONS: This study showed a significant, under-utilised potential for improving GPs' awareness about children of parents with depression. Perceived knowledge of the potential impact of parental depression was crucial for the attention given to the children. FUNDING: The study was funded by The Central Denmark Region and the Danish National Research Foundation for Primary Care. TRIAL REGISTRATION: not relevant.

Structures of insect Imp-L2 suggest an alternative strategy for regulating the bioavailability of insulin-like hormones.

The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.

Structure of rat acidic fibroblast growth factor at 1.4 A resolution.

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related heparin-binding polypeptides that are involved in the regulation of cell growth, survival, differentiation and migration. Here, a 1.4 A resolution X-ray structure of rat FGF1 is presented. Two molecules are present in the asymmetric unit of the crystal and they coordinate a total of five sulfate ions. The structures of human, bovine and newt FGF1 have been published previously. Human and rat FGF1 are found to have very similar structures.

Thyroid function during B-vitamin supplementation of patients on antiepileptic drugs.

OBJECTIVES: Patients on antiepileptic drugs (AEDs) may have low serum concentrations of thyroxine, with or without a compensatory increment in thyroid-stimulating hormone (TSH). Furthermore, patients on AEDs often have hyperhomocysteinemia and low concentrations of vitamins B(6), B(2) and folate. Previously, an inverse relationship between thyroxine and homocysteine concentrations has been observed. In animals, deficiency of vitamin B(6) has been found to impair the hypophyseal release of TRH. We have studied the effect of B-vitamin supplements on thyroid function in patients on AEDs. DESIGN AND METHODS: Thirty-two patients on AEDs were identified with hyperhomocysteinemia and low folate, B(6) and B(2). They were supplemented with pyridoxine, riboflavin and folic acid for 30 days. RESULTS: At baseline, the patients had low serum concentrations of free thyroxin and slightly elevated TSH. On day 30 of the B-vitamin supplements, homocysteine had decreased, however, the thyroid parameters remained unchanged. CONCLUSIONS: Hyperhomocysteinemic patients on AEDs have indications of hypothyroidism, however, supplementation with B-vitamins does not improve their thyroid function.

Chaperone binding at the ribosomal exit tunnel.

The exit tunnel region of the ribosome is well established as a focal point for interaction between the components that guide the fate of nascent polypeptides. One of these, the chaperone trigger factor (TF), associates with the 50S ribosomal subunit through its N-terminal domain. Targeting of TF to ribosomes is crucial to achieve its remarkable efficiency in protein folding. A similar tight coupling to translation is found in signal recognition particle (SRP)-dependent protein translocation. Here, we report crystal structures of the E. coli TF ribosome binding domain. TF is structurally related to the Hsp33 chaperone but has a prominent ribosome anchor located as a tip of the molecule. This tip includes the previously established unique TF signature motif. Comparison reveals that this feature is not found in SRP structures. We identify a conserved helical kink as a hallmark of the TF structure that is most likely critical to ensure ribosome association.

The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity.

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.

Clinical importance of neutralising antibodies against interferon beta in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Interferon beta is the first-line treatment for relapsing-remitting multiple sclerosis, but the drug can induce neutralising antibodies against itself, which might reduce effectiveness. We aimed to assess the clinical effect of neutralising antibodies. METHODS: We measured neutralising antibodies every 12 months for up to 60 months in 541 patients with multiple sclerosis, randomly selected from all patients who started treatment with interferon beta between 1996 and 1999. Patients left the study if they changed or discontinued therapy. Antibodies were measured blindly, using antiviral neutralisation bioassays with high, medium, and low sensitivity, and with different neutralising capacities as cutoff value for definition of a neutralising-antibody-positive result. FINDINGS: Patients developed neutralising antibodies independent of age, sex, disease duration, and progression index at start of treatment. Relapse rates were significantly higher during antibody-positive periods (0.64-0.70) than they were during antibody-negative periods (0.43-0.46; p<0.03). When comparing the number of relapses in the neutralising-antibody-positive and neutralising-antibody-negative periods we found odds ratios in the range 1.51 to 1.58 (p<0.03). Time to first relapse was significantly increased by 244 days in patients who were antibody-negative at 12 months (log rank test 6.83, p=0.009). During this short-term study, presence of neutralising antibodies did not affect disease progression measured with the expanded disability status scale. INTERPRETATION: Our findings suggest that the presence of neutralising antibodies against interferon beta reduces the clinical effect of the drug. In patients who are not doing well on interferon beta, the presence of such antibodies should prompt consideration about change of treatment.

Expression, crystallization and preliminary X-ray analysis of the extracellular Ig modules I-IV and F3 modules I-III of the neural cell-adhesion molecule L1.

Four amino-terminal immunoglobulin (Ig) modules and three fibronectin type III (F3) modules of the mouse neural cell-adhesion molecule L1 have been expressed in Drosophila S2 cells. The Ig modules I-IV of L1 crystallized in a trigonal space group, with unit-cell parameters a = b = 239.6, c = 99.3 A, and the crystals diffracted X-rays to a resolution of about 3.5 A. The F3 modules I-III of L1 crystallized in a tetragonal space group, with unit-cell parameters a = b = 80.1, c = 131 A, and the crystals diffracted X-rays to 2.8 A resolution. This is a step towards the structure determination of the multimodular constructs of the neural cell-adhesion molecule L1 in order to understand the function of L1 on a structural basis.

Structure and binding properties of a cameloid nanobody raised against KDM5B.

The histone demethylase KDM5B is considered to be a promising target for anticancer therapy. Single-chain antibodies from llama (nanobodies) have been raised to aid in crystallization and structure determination of this enzyme. The antigen-binding properties of 15 of these nanobodies have been characterized. The crystal structure of one of these (NB17) has been determined to a resolution of 1.85 Å. NB17 crystallizes in space group P4322 with six molecules in the asymmetric unit. The six molecules in the asymmetric unit pack as an entity with approximate D3 symmetry with interactions mediated by the CDR loops, which could act as a crystallization nucleus. NB17 does not bind to monomeric KDM5B residues 1-820, but is found to bind to aggregates formed after incubation at 310 K.

Cessation of smoking after first-ever stroke: a follow-up study.

BACKGROUND AND PURPOSE: Cessation of smoking is widely recommended in patients with stroke to reduce the risk of myocardial infarction and recurrent stroke, but little is known regarding how patients modify their smoking habits after a stroke. We used data from a prospective follow-up study to assess modification of smoking habits and to identify predictors of persistent smoking after first-ever stroke. METHODS: All patients admitted to the only neurology department of Funen County (465 000 inhabitants) with first-ever stroke from August 1, 1999, to January 31, 2001, were prospectively identified. A comprehensive structured interview was completed both during hospitalization and at 6-month follow-up. The interview comprised questions on education, occupation, marital status, lifestyle, concomitant diseases, and functional disability. We estimated the relative risk of persistent smoking at follow-up using unconditional logistic regression. RESULTS: We identified 734 patients with a first-ever stroke in the study period. One hundred three patients (14%) died in the 6-month period after their admission. A total of 511 patients (81%) who participated in the interview both on admission and at follow-up were included in the present study. Among 198 patients (38.7%) who were current smokers on admission, 43 patients (21.7%) gave up smoking within 6 months of suffering a stroke. Sex, functional status, and sociodemographic characteristics were independently associated with persistent smoking. CONCLUSIONS: Our results suggest that more efficient antismoking counseling is required to reduce the proportion of persistent smokers after stroke. This counseling should take into account the increased risk of persistent smoking in men, patients with no disability, blue-collar workers, and patients living alone.

Is tRNA binding or tRNA mimicry mandatory for translation factors?

tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

Crystal structures of the human G3BP1 NTF2-like domain visualize FxFG Nup repeat specificity.

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P212121 and P6322 based on two different constructs, residues 1-139 and 11-139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P212121 crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design.