New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

The solution structure of Co‚Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) complexed with the self-complementary oligonucleotide d(CCAGGCCTGG) with a cleavage site at C6 has been determined by 2D NMR spectroscopic methods and molecular dynamics calculations. Intermolecular NOEs (60 between CoBLM A2 green and DNA) and intramolecular NOEs (61 within CoBLM A2 green) have defined the position and orientation of CoBLM A2 green with respect to its single binding site in the duplex. CoBLM A2 green is a stable analog of the activated BLM, the Fe 3+ hydroperoxide (Sam, J. W.; Tang, X.-J.; Peisach, J. J. Am. Chem. Soc. 1994, 116, 5250-5256). These studies have provided the first structural insight into the mode of binding of the bithiazole tail of CoBLM A2 green to DNA, the basis for specificity of its cleavage at pyrimidines (Py) in d(G-Py) sequences, and the orientation of its terminal oxygen of the hydroperoxide relative to the 4′ carbon hydrogen bond being cleaved in the DNA. The bithiazole tail inserts 3′ to the C6 cleavage site from the minor groove. The terminal thiazolium ring is completely stacked between the bases of G14 and G15, while the penultimate thiazolium ring is only partially stacked between the bases of C6 and C7. The bithiazole tail thus binds via a partial intercalation mode and the DNA is unwound by 13°over the (G5‚C16)∼(C6‚G15)∼(C7‚G14)∼(T8‚A13) steps. No specific interactions between the bithiazole tail and the DNA have been identified, and thus, this interaction does not define the BLM's cleavage specificity but its binding affinity. The metal binding domain and the peptide linker region of CoBLM A2 green bind within the minor groove of the duplex and define the basis for its specificity of DNA cleavage. The 4-amino group and the N3 of the pyrimidine ring of CoBLM A2 green form specific hydrogen bonds with the N3 and the 2-amino group, respectively, of the G5 in the duplex and provide an unusual example of a minor groove base triplelike interaction. A basis for the preference for G over A, 5′ to the Py cleavage site, is thus established. The metal binding domain and the valeryl moiety in the linker have a conformation strikingly similar to that defined in the free CoBLM A2 green (Wu, W.; Vanderwall, D. E.; Lui, S. M.; Tang, X.-J.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. ). The most remarkable feature of this structure is the observation of the proton associated with the hydroperoxide of CoBLM A2 green and its observed intermolecular NOEs to the minor groove protons of C6 and C7 of the duplex. Thus this structure provides a rare snapshot of an analog of a reactive intermediate poised to initiate the hydrogen atom abstraction event. The molecular modeling reveals that the distal oxygen of the hydroperoxide is 2.5 Å from the 4′-hydrogen of C6. A number of additional intramolecular hydrogen bonds between the hydroperoxide ligand and the peptide linker region are also proposed, which appear to play a key role in positioning the reactive intermediate near the hydrogen atom being abstr...

The structure of homogeneous Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) has been determined using 2D NMR methods and molecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907−916) reported several possible structures for CoBLM A2 green compatible with their NMR data acquired on a mixture of CoBLM A2 green and A2 brown forms. The availability of the pure CoBLM A2 green, which is stable for months at neutral pH, has allowed the complete assignments of the 1H and 13C chemical shifts, observation of 55 intramolecular NOEs, and determination of 15 coupling constants allowing the definition of dihedral angles. These results are a prerequisite to determining its structure with duplex DNA of a defined sequence (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). Two screw sense isomers each containing two possible axial ligands (the primary amine of the β-aminoalanine and the carbamoyl nitrogen of the mannose) were considered as viable candidates for the structure of CoBLM A2 green. Using the NMR constraints and molecular dynamics calculations, the structures of all four isomers were generated. One set of screw sense isomers can be readily eliminated from considerations based on violations of NOE and dihedral angle constraints. The other screw sense isomer containing either one or the other of the postulated axial ligands has been examined in some detail. The structure containing the primary amine of β-aminoalanine as the axial ligand is favored on the basis of coupling constants and NOE arguments, potential energy considerations, model studies, and studies with analogs of BLM. The favored structure is compact with the bithiazole moiety folded back underneath the equatorial plane of the metal binding domain, on the same face as the hydroperoxide ligand. The geometry of the peptide linker is very well defined by the observed coupling constants in the valeryl and threonine moieties of the linker. CoBLM A2 green has been studied with two self-complementary oligonucleotides, d(CCAGGCCTGG) and d(CCAGTACTGG). Both of these oligomers possess a single, UV light-mediated cleavage site (C and T, respectively). In addition, fluorescent quenching studies have allowed the determination of the first sequence-specific dissociation constants of 1.7 × 10-7 and 1.5 × 10-7 M, respectively. Titration of CoBLM A2 green with each of these oligomers reveals a 1:1 complex in slow exchange on the NMR time scale. The upfield shifts of the bithiazole protons in both of these complexes are indicative of a partial intercalative mode of binding. The stage is now set for the determination of the structure of the CoBLM A2 green bound sequence specifically to DNA.