Three-dimensional structure of a recombinant gap junction membrane channel.
(1/1562)
Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu. (+info)
Structure of DNA-dependent protein kinase: implications for its regulation by DNA.
(2/1562)
DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described. (+info)
Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.
(3/1562)
Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs. (+info)
Biochemical evolution III: polymerization on organophilic silica-rich surfaces, crystal-chemical modeling, formation of first cells, and geological clues.
(4/1562)
Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources. Crystal-chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite). Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al-OH site per 25 neutral Si tetrahedral sites. Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species. Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms. The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich "soup." Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments. Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones. All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species. Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments. (+info)
Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling.
(5/1562)
The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208. (+info)
Molecular dynamics simulation of alpha-lactalbumin and calcium binding c-type lysozyme.
(6/1562)
Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at the bottom of the cleft and is important for the structural integrity of the protein. Calcium is an ubiquitous intracellular signal in higher eukaryotes and structural changes induced on calcium binding have been observed in a number of proteins. In the present study, molecular dynamics simulations of equine LYZ and human LA, with and without calcium, were carried out. We detail the differences in the dynamics of equine LYZ and human LA, and discuss it in the light of experimental data already available and relate it to the behavior of the functionally important regions of both the proteins. These simulations bring out the role of calcium in the conformation and dynamics of these metallo-proteins. In the calcium bound LA, the region of the protein around the calcium binding site is not only frozen but the atomic fluctuations are found to increase away from the binding site and peak at the exposed sites of the protein. This channeling of fluctuations away from the metal binding site could serve as a general mechanism by which the effect of metal binding at a site is transduced to other parts of the protein and could play a key role in protein-ligand and/or protein-protein interaction. (+info)
Homogenization and crystallization of histidine ammonia-lyase by exchange of a surface cysteine residue.
(7/1562)
Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonas putida was expressed in Escherichia coli and purified. In the absence of thiols the tetrameric enzyme gave rise to undefined aggregates and suitable crystals could not be obtained. The solvent accessibility along the chain was predicted from the amino acid sequence. Among the seven cysteines, only one was labeled as 'solvent-exposed'. The exchange of this cysteine to alanine abolished all undefined aggregations and yielded readily crystals diffracting to 1.8 A resolution. (+info)
Iron in the basal ganglia in Parkinson's disease. An in vitro study using extended X-ray absorption fine structure and cryo-electron microscopy.
(8/1562)
Iron is found in high concentration in some areas of the brain, and increased iron in the substantia nigra is a feature of Parkinson's disease. The purpose of this study was to investigate the physical environment of brain iron in post-mortem tissue to provide information on the possible role of iron in neurodegeneration in Parkinson's disease. Iron has also been implicated as the cause of signal loss in areas of high brain iron on T2-weighted MRI sequences. Knowledge of the physical environment of the brain iron is essential in interpreting the cause of signal change. Post-mortem tissue was obtained from six cases of Parkinson's disease and from six age-matched controls. Iron levels were measured using absorption spectrophotometry. Extended X-ray absorption fine structure was used to evaluate the atomic environment of iron within the substantia nigra and both segments of the globus pallidus. Cryo-electron transmission microscopy was used to probe the iron storage proteins in these areas. Iron levels were increased in the parkinsonian nigra and lateral portion of the globus pallidus. Spectra from the extended X-ray absorption fine structure experiments showed that ferritin was the only storage protein detectable in both control and parkinsonian tissue in all areas studied. Cryo-electron transmission microscopy studies showed that ferritin was more heavily loaded with iron in Parkinson's disease when compared with age-matched controls. In summary we have shown that iron levels are increased in two areas of the brain in Parkinson's disease including the substantia nigra, the site of maximal neurodegeneration. This produces increased loading of ferritin, which is the normal brain iron storage protein. It is possible that increased loading of ferritin may increase the risk of free radical-induced damage. Differences in ferritin loading may explain regional differences in iron's effect on the T2 signal. (+info)