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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers. A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain. The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III. Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer. The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.
J Mol Biol 1997 Jul 04
PMID:Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. 923 98

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.
J Mol Biol 1998 Jan 09
PMID:The solution structure of functionally active human proliferating cell nuclear antigen determined by small-angle neutron scattering. 945 44

The crystal structure of a phosphonate complex of the class A TEM-1 beta-lactamase has been determined to a resolution of 2.0 A. The phosphonate appears stoichiometrically at the active site, bound covalently to Ser70Ogamma, with one phosphonyl oxygen in the oxyanion hole. Although the overall structure is very similar to that of the native enzyme (rms difference 0.37 A for all heavy atoms), changes have occurred in the position of active site functional groups. The active site is also not in the conformation observed in the complex of another class A beta-lactamase, that of Staphylococcus aureus PC1, with the same phosphonate [Chen, C. C. H., et al. (1993) J. Mol. Biol. 234,165-178]. Both phosphonate structures, however, can be seen to represent models of acylation transition-states since in each the deacylating water molecule appears firmly bound to the Glu166 carboxylate group. The major difference between the structures lies in the positioning of Lys73Nzeta and Ser130Ogamma. In the S. aureus structure, the closest interaction of these functional groups is between Lys73Nzeta and Ser70Ogamma (2.8 A), while in the TEM-1 structure it is between Ser130Ogamma and the second phosphonyl oxygen of the bound inhibitor (2.8 A). The former structure therefore may resemble a transition state for formation of the tetrahedral species in acylation by nucleophilic attack on the substrate, where Lys73Nzeta presumably catalyzes the reaction as a general base. The TEM-1 structure can then be seen as an analogue of the transition state for breakdown of the tetrahedral species, where Ser130Ogamma is acting as a general acid, assisting the departure of the leaving group. The class A beta-lactamase crystal structures now available lead to a self-consistent proposal for a mechanism of catalysis by these enzymes.
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PMID:Crystal structure of an acylation transition-state analog of the TEM-1 beta-lactamase. Mechanistic implications for class A beta-lactamases. 948 12

Bacterial flagellum has a cap structure tightly attached to its distal end. The cap is an oligomeric assembly of HAP2 protein (also called FliD) and plays an essential role in the filament growth in vivo by preventing flagellin monomers from leaking out without polymerization. Electron micrographs of the HAP2 complex formed in solution showed exclusively a pentagonal shape, called "star-cap", which was thought to be the end-on view of the cap. The molecular mass roughly corresponded to a dodecamer of HAP2, and therefore a double-layered star-cap was modeled to be the cap. Here, we have observed the side view of the complex in electron micrographs. The images clearly show a rectangular shape, about 80 A wide and 180 A long, with a bipolar feature in its long axis, indicating that the complex is a bipolar pair of pentamers. A thin plate feature is identified at each end of the particle, which looks exactly like the one observed as the structure of the native filament cap. Together with the structure of the filament previously analyzed by electron cryomicroscopy, the results suggest that the cap is a pentamer with its thin plate exposed to the solvent and the other half plugged into the hole at the distal end of the filament, which is almost twice wider than its central channel. This also allows us to model the axial domain arrangement of flagellin subunit in the filament.
J Mol Biol 1998 Apr 10
PMID:Plugging interactions of HAP2 pentamer into the distal end of flagellar filament revealed by electron microscopy. 954 71

We tested four hypotheses about the relationships of the kinglets (genus Regulus)to seven closely related genera of the songbird superfamily Sylvioidea using mitochondrial DNA sequences. The kinglets were suggested to be closely related to the tits (Parus) or to the Old World Warblers (Phylloscopus) and were also suggested to constitute the, or at least one of the, most ancestral splits among the sylvioids. Our phylogenetic analysis grouped the kinglets as the sister group of a clade comprising Parus and Phylloscopus and including the genera Sylvia, Aegithalos, and Leptopoecile. Two of the taxa were placed more ancestral to the kinglets: Sitta and Certhia. We also identified the endemic kinglet species from the Canary Islands s the sister group of R. regulus. The superimposition of breeding behavior on the phylogeny suggests that hole nesting is ancestral and various other patterns of nest construction have evolved from it. The placement of Parus implies that hole nesting in the Paridae is likely to have originated secondarily. Further, Leptopoecile and Aegithalos, two genera for which a helper system of elder offspring in breeding was described, were resolved as a clade.
Mol Phylogenet Evol 1998 Aug
PMID:Mitochondrial phylogeny of the genus Regulus and implications on the evolution of breeding behavior in sylvioid songbirds. 975 24

The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication. The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate. We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein. Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views. Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture. These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole. A clear structural handedness has also been detected in some of these forms. An analysis of these quaternary states and a model for the structural organization of G40P are presented.
J Mol Biol 1998 Nov 06
PMID:Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P). 979 Aug 42

A comparison has been made for the quenching effect of three classes of organic solvents on the porous silicon photoluminescence, including hydrocarbons, solvents containing oxygen and nitrogen. Among them organoamines possess the strongest quenching effect. The experiments show that the solvent molecules interact synergistically with porous silicon and the main pathways include the surface hydrophobic interaction, dipole-dipole interaction and hole trapping, the later being the dominant pathway in quenching of porous silicon photoluminescence.
Spectrochim Acta A Mol Biomol Spectrosc 1998 Sep
PMID:Organic solvent induced quenching of porous silicon photoluminescence. 980 39

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.
J Mol Biol 1998 Dec 11
PMID:Visualisation of human rad52 protein and its complexes with hRad51 and DNA. 983 24

Several experimental data support the notion that the recognition of DNA crossovers play an important role in the multiple functions of topoisomerase II. Here, a theoretical analysis of the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers is performed, using the crystal coordinates of the docking partners. The DNA crossovers are assumed to be clamped into the central hole of the enzyme. Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein-DNA interactions, this analysis shows that two geometric solutions could exist, depending on the chirality of the DNA crossovers. In the first one, the two DNA segments are symmetrically recognized by the enzyme while each single double helix binds asymmetrically the protein dimer. In the second one, each double helix is symmetrically recognized by the protein around its dyad axis, while the two DNA segments have their own binding modes. The finding of potential DNA-binding domains which could interact with the crossovers provides structural supports for each model. The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA-binding site of histone H5 which binds DNA crossover is of particular interest. Each solution, which is consistent with different sets of experimental data found in the literature, could either correspond to different functions of the enzyme or different steps of the reaction. This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes, and raises new questions about the relationships between the mechanism of strand-passage and strand-exchange catalyzed by the enzyme.
J Mol Biol 1998 Dec 18
PMID:Symmetry and chirality in topoisomerase II-DNA crossover recognition. 987 50

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix. In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix. The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.
J Mol Biol 1999 Jan 29
PMID:Crystal structure of CcdB, a topoisomerase poison from E. coli. 991 4


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