Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.

Bathorhodopsin structure in the room-temperature rhodopsin photosequence: picosecond time-resolved coherent anti-Stokes Raman scattering.

Structural changes in the retinal chromophore during the formation of the bathorhodopsin intermediate (bathoRT) in the room-temperature rhodopsin (RhRT) photosequence (i.e., vision) are examined using picosecond time-resolved coherent anti-Stokes Raman scattering. Specifically, the retinal structure assignable to bathoRT following 8-ps excitation of RhRT is measured via vibrational Raman spectroscopy at a 200-ps time delay where the only intermediate present is bathoRT. Significant differences are observed between the C=C stretching frequencies of the retinal chromophore at low temperature where bathorhodopsin is stabilized and at room temperature where bathorhodopsin is a transient species in the RhRT photosequence. These vibrational data are discussed in terms of the formation of bathoRT, an important step in the energy storage/transduction mechanism of RhRT.

Subparsec-scale structure and evolution of Centaurus A (NGC5128).

We present a series of 8.4-GHz very-long-baseline radio interferometry images of the nucleus of Centaurus A (NGC5128) made with a Southern Hemisphere array, representing a 3.3-year monitoring effort. The nuclear radio jet is approximately 50 milliarcseconds in extent, or at the 3.5-megaparsec distance of NGC5128, approximately 1 parsec in length. Subluminal motion is seen and structural changes are observed on time scales shorter than 4 months. High-resolution observations at 4.8 and 8.4 GHz made in November 1992 reveal a complex morphology and allow us to unambiguously identify the self-absorbed core located at the southwestern end of the jet.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Receptive field structure in the visual cortex: does selective stimulation induce plasticity?

Sensory areas of adult cerebral cortex can reorganize in response to long-term alterations in patterns of afferent signals. This long-term plasticity is thought to play a crucial role in recovery from injury and in some forms of learning. However, the degree to which sensory representations in primary cortical areas depend on short-term (i.e., minute to minute) stimulus variations remains unclear. A traditional view is that each neuron in the mature cortex has a fixed receptive field structure. An alternative view, with fundamentally different implications for understanding cortical function, is that each cell's receptive field is highly malleable, changing according to the recent history of the sensory environment. Consistent with the latter view, it has been reported that selective stimulation of regions surrounding the receptive field induces a dramatic short-term increase in receptive field size for neurons in the visual cortex [Pettet, M. W. & Gilbert, C. D. (1992) Proc. Natl. Acad. Sci. USA 89, 8366-8370]. In contrast, we report here that there is no change in either the size or the internal structure of the receptive field following several minutes of surround stimulation. However, for some cells, overall responsiveness increases. These results suggest that dynamic alterations of receptive field structure do not underlie short-term plasticity in the mature primary visual cortex. However, some degree of short-term adaptability could be mediated by changes in responsiveness.

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.