Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Equilibrium distributions of topological states in circular DNA: Interplay of supercoiling and knotting

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and ΔLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(ΔLk, K), is related to two conditional distributions: P(ΔLk|K), the distribution of ΔLk for a particular K, and P(K|ΔLk) and also to two simple distributions: P(ΔLk), the distribution of ΔLk irrespective of K, and P(K). We explored the relationships between these distributions. P(ΔLk, K), P(ΔLk), and P(K|ΔLk) were calculated from the simulated distributions of P(ΔLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|ΔLk), the distribution of knot types for a particular value of ΔLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of ΔLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |ΔLk| > 12, only one or two knot types dominate the P(K|ΔLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides.

In tetrapods, only one gene encoding a somatostatin precursor has been identified so far. The present study reports the characterization of the cDNA clones that encode two distinct somatostatin precursors in the brain of the frog Rana ridibunda. The cDNAs were isolated by using degenerate oligonucleotides based on the sequence of the central region of somatostatin to screen a frog brain cDNA library. One of the cDNAs encodes a 115-amino acid protein (prepro-somatostatin-14; PSS1) that exhibits a high degree of structural similarity with the mammalian somatostatin precursor. The other cDNA encodes a 103-amino acid protein (prepro-[Pro2, Met13]somatostatin-14; PSS2) that contains the sequence of the somatostatin analog (peptide SS2) at its C terminus, but does not exhibit appreciable sequence similarity with PSS1 in the remaining region. In situ hybridization studies indicate differential expression of the PSS1 and PSS2 genes in the septum, the lateral part of the pallium, the amygdaloid complex, the posterior nuclei of the thalamus, the ventral hypothalamic nucleus, the torus semicircularis and the optic tectum. The somatostatin variant SS2 was significantly more potent (4-6 fold) than somatostatin itself in displacing [125I-Tyr0, D-Trp8] somatostatin-14 from its specific binding sites. The present study indicates that the two somatostatin variants could exert different functions in the frog brain and pituitary. These data also suggest that distinct genes encoding somatostatin variants may be expressed in the brain of other tetrapods.

Active site topology and reaction mechanism of GTP cyclohydrolase I.

GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.

The case for unification.

I investigate the issue of whether the various subclasses of radio-loud galaxies are intrinsically the same but have been classified differently mainly due to their being viewed from different directions. Evidence for the two key elements of this popular version of the "unified scheme (US)," relativistic jets and nuclear tori, is updated. The case for the torus opening angle increasing with the radio luminosity of the active galactic nucleus (AGN) is freshly argued. Radio-loud AGN are particularly suited for testing the US, since their structures and polarization properties on different scales, as well as their overall radio sizes, provide useful statistical indicators of the relative orientations of their various subclasses. I summarize recent attempts to bring under a single conceptual framework the USs developed for radio-moderate [Fanaroff-Riley type I (FRI)] and radio-powerful (FRII) AGN. By focusing on FRII radio sources, I critically examine the recent claims of conflict with the US, based on the statistics of radio-size measurements for large, presumably orientation-independent, samples with essentially complete optical identifications. Possible ways of reconciling these results, and also the ones based on very-long-baseline radio interferometry polarimetric observations, with the US are pointed out. By incorporating a highly plausible temporal evolution of radio source properties into the US, I outline a scenario that allows the median linear size of quasars to approach, or even exceed, that of radio galaxies, as samples with decreasing radio luminosity are observed. Thus, even though a number of issues remain to be fully resolved, the scope of unified models continues to expand.

Difficulties with unification.

The difficulties perceived in the orientation-based unified scheme models, when confronted with the observational data, are pointed out. It is shown that in meter-wavelength selected samples, which presumably are largely free of an orientation bias, the observed numbers of quasars versus radio galaxies are not in accordance with the expectations of the unified scheme models. The observed number ratios seem to depend heavily on the redshift, fluxdensity, or radio luminosity levels of the selected sample. This cannot be explained within the simple orientation-based unified scheme with a fixed average value of the half-opening angle (c approximately 45 degrees ) for the obscuring torus that supposedly surrounds the nuclear optical continuum and the broad-line regions. Further, the large differences seen between radio galaxies and quasars in their size distributions in the luminosity-redshift plane could not be accommodated even if I were to postulate some suitable cosmological evolution of the opening angle of the torus. Some further implications of these observational results for the recently proposed modified versions of the unified scheme model are pointed out.