Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A.

Gene transcription requires the release of inactive DNA from its packaging of histone proteins. Following the discovery of the first transcription-associated histone acetyltransferase, tetrahymena GCN5, it was shown that yeast GCN5 is recruited to the promoter and causes hyper-acetylation of histones and transcriptional activation of target genes, establishing a direct connection between histone acetylation and transcriptional activation. Many other important transcription regulators have been found to have histone acetyltransferase activity, including TAFII230/250, p300/CBP and its associated factor PCAF. Here we present the solution structure of the catalytic domain of tGCN5 (residues 47-210) in complex with coenzyme A. The structure contains two domains; the amino-terminal domain is similar to those of other GCN5-related N-acetyltransferases but the carboxy-terminal domain is not. Coenzyme A binds in a deep hydrophobic pocket between the two domains. Chemical shift changes upon titration with histone H3 peptides indicate a binding site at the domain boundary opposite to the coenzyme A site. The structural data indicate a single-step acetyl-transfer reaction mechanism catalysed by a hydrogen bond to the backbone amide group of leucine 126 and the side-chain carboxyl group of a conserved acidic residue.

Structure and interactions of the translation initiation factor eIF1.

eIF1 is a universally conserved translation factor that is necessary for scanning and involved in initiation site selection. We have determined the solution structure of human eIF1 with an N-terminal His tag using NMR spectroscopy. Residues 29-113 of the native sequence form a tightly packed domain with two alpha-helices on one side of a five-stranded parallel and antiparallel beta-sheet. The fold is new but similar to that of several ribosomal proteins and RNA-binding domains. A likely binding site is indicated by yeast mutations and conserved residues located together on the surface. No interaction with recombinant eIF5 or the initiation site RNA GCCACAAUGGCA was detected by NMR, but GST pull-down experiments show that eIF1 binds specifically to the p110 subunit of eIF3. This interaction explains how eIF1 is recruited to the 40S ribosomal subunit.

The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein.

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ¿1H¿-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ¿1H¿-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.

4E binding proteins inhibit the translation factor eIF4E without folded structure.

The 4E binding proteins (4E-BP1 and 4E-BP2) inhibit translation by binding to the limiting, proto-oncogenic initiation factor eIF4E. 4E-BPs produced in Escherichia coli had little or no folded structure, measured by NMR and CD. However, these proteins inhibited translation in reticulocyte lysate. Furthermore, they bound to isolated mouse eIF4E, showing a few broader, dispersed new NMR signals but no general increase in chemical shift dispersion. A peptide with the sequence of 4E-BP1 residues 49-68 was sufficient to bind eIF4E and to inhibit translation in reticulocyte lysate. These results suggest that a short central region of the 4E-BPs is responsible for eIF4E binding and translation inhibition while the remainder is unfolded and flexible.

Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein.

eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.

Inuit attitudes toward deviant behavior: a vignette study.

Attitudes toward deviant behavior that might indicate psychiatric disorder were investigated among the Inuit of Northern Québec (Nunavik). In a convenience sample of 137 Inuit adults, respondents were randomly presented with one of six different vignettes that described a man with "strange" behavior who was either threatening or withdrawn and whose problem was labeled either "isumaluttuq" ("burdened or weighed down by thoughts"), "demon possession," or "mental illness." Respondents rated their willingness to live, work, or hunt with this person and allow him into their family on a social distance scale. Significant predictors of greater social distance were female gender, more education, less familiarity with the behavior, and perception of the person as less likely to recover. There were no significant effects of vignette behavior or label on social distance ratings. Rating of likelihood of recovery was influenced by the vignette label, with isumaluttuq associated with less chance of recovery. Ascribing strange behavior to morally wrong action and to spirits or demons were highly inter-correlated and each was associated with perception of greater likelihood of recovery. Results suggest that Inuit attitudes toward deviant behavior are influenced more by perceived familiarity and likelihood of recovery than by labels, causal attributions, or explanations. The indigenous psychological concept of isumaluttuq does not serve to reduce social stigma. Efforts to promote the community integration of psychiatric patients through education should aim to increase familiarity with the problematic behavior and emphasize potential for recovery.

Treatment of NOE constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures.

Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for 'pseudoatom corrections' is proposed for use with calculations employing 'centre-averaging'. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using 'r(-6) averaging' results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using 'r(-6) summation' can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r(-6) averaging, r(-6) summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r(-6) averaging or r(-6) summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.

Structure of a soluble, glycosylated form of the human complement regulatory protein CD59.

BACKGROUND: CD59 is a cell-surface glycoprotein that protects host cells from complement-mediated lysis by binding to and preventing the normal functioning of the complement proteins C8 and/or C9 which form part of a membrane penetrating assembly called the membrane attack complex. CD59 has no structural similarity to other complement proteins, but is an example of a plasma protein domain type found also in murine Ly-6 proteins and the urokinase-type plasminogen activator receptor. RESULTS: CD59 was purified from human urine, retaining the N-glycan and at least some of the non-lipid component of the glycosylphosphatidylinositol membrane anchor. The three-dimensional structure of the protein component has been determined in the presence of the carbohydrate groups using two-dimensional NMR spectroscopy. The protein structure is well defined by the NMR data (root mean square deviation from the mean structure of 0.65 A for backbone atoms and no distance constraint violations greater than 0.4 A). Structure calculations were also carried out to model the orientation of the N-acetylglucosamine residue that is directly linked to Asn18. CONCLUSIONS: The main features of the protein structure are two antiparallel beta-sheets (a central one with three strands and another with two), a short helix that packs against the three-stranded beta-sheet, and a carboxy-terminal region that, although lacking regular secondary structure, is well defined and packs against the three-stranded beta-sheet, on the opposite face to the helix. We have used the structure, in combination with existing biochemical data, to identify residues that may be involved in C8 binding.

Sequence-specific 1H-NMR assignments and folding topology of human CD59.

CD59 is a recently discovered cell-surface glycoprotein that restricts lysis by homologous complement and has limited sequence similarity to snake venom neurotoxins. This paper describes the first results of a two-dimensional NMR study of CD59 prepared from human urine. Nearly complete 1H-NMR assignments were obtained for the 77 amino acid residues and partial assignments for the N-glycan and the glycosylphosphatidylinositol (GPI) anchor. These results together confirm that the C-terminal residue of the mature protein is Asn 77 and that the urine-derived form retains the nonlipid part of the GPI anchor. The data further indicate that the GPI anchor and possibly the N-glycan are structurally inhomogeneous and suggest that the phospholipid present in the intact GPI anchor was removed by phosphatidylinositol-specific phospholipase-D. The folding topology of the protein was determined from NOE enhancements and slowly exchanging backbone amide protons and consists primarily of five extended strands (denoted beta 1-beta 5 in sequence order), arranged into separate two-stranded (beta 1 and beta 2) and three-stranded (beta 3-beta 5) antiparallel beta-sheets. The same folding topology is found in all of the snake venom neurotoxins whose structures have been determined. The region between the beta 4 and beta 5 strands has helical character, a feature that is not present in the neurotoxins but that is seen in the topologically similar wheat germ agglutinin.

The normocomplementemic urticarial vasculitis syndrome--report of a case and response to colchicine.

A patient with a 15-year history of urticarial vasculitic rashes and chronic vasculitic skin ulceration has been followed at our Connective Tissue Disease Clinic for the past five years. Serum complement levels have been persistently normal. She has been unresponsive to a variety of medications including steroids, dapsone and hydroxychloroquine. She had a dramatic response to colchicine 500 micrograms daily with total clearing of the urticarial vasculitic rash.

The rule learning behavior of reading disabled and normal children as a function of task characteristics and instruction.

The purpose of this research was to investigate rule learning in reading disabled (RD) and normal children (chronological age and reading age (RA) match) when required to (a) abstract rules independently, and (b) use rules after instruction. Study 1 required the children to solve problems using shapes and letters. Although there was no difference between groups in the rate of problem solving when children were asked to abstract rules independently, the pattern of errors was different. The RD children made a greater proportion of errors on the negative instance for the more complex problems. In particular, this occurred on the letter task which involved psycholinguistic categorization. After instruction, the RA controls made more errors than the other groups. Study 2 was an analogous pseudoword reading task. Even with statistical adjustment for differences in prior grapheme-phoneme (g-p) rule knowledge, the RD children performed less accurately than the RA controls when they had to abstract rules, although this was restricted to the most difficult rule (rule of e). There was no difference after instruction in rule application, although the pattern of errors and post-test results indicated that the RD children continued to experience decoding difficulty. These results suggest a phonologically based productive deficit which interferes with the learning of g-p rules. This may be part of a more general language deficit which includes psycholinguistic categorization. Despite the severity of this handicap, RD children seem responsive to instruction.

The relevance in adults of air-flow obstruction, but not of mucus hypersecretion, to mortality from chronic lung disease. Results from 20 years of prospective observation.

From 1954 to 1961, pulmonary function was assessed in 2,718 British men by forced expiratory maneuvers, and mucus hypersecretion and smoking habits were assessed by questionnaires. In 20 to 25 yr of follow-up, 104 men (all of whom had smoked) died of chronic obstructive pulmonary disease (COPD). The risk of death from COPD was strongly correlated with the initial degree of air-flow obstruction. Among men with similar initial air-flow obstruction, however, age-specific COPD death rates were not significantly related to initial mucus hypersecretion, supporting the concept that air-flow obstruction and mucus hypersecretion are largely independent disease processes. A moderate relationship existed between initial mucus hypersecretion and subsequent lung cancer mortality, but it is not known whether this was due solely to a common correlation of both conditions with the effective degree of exposure of the large bronchi to causative factors such as tobacco smoke.