RESUMEN
We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting P(IB)-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH metal-binding motif in TM6 and a histidine-rich N-terminal metal-binding domain. We have cloned both copper pumps, expressed them in Escherichia coli and characterized them functionally. CtrA2 is activated by Ag(+) and Cu(+) and presumably transports reduced Cu(+), while CtrA3 is activated by, and presumably transports, the oxidized copper ion. Both CtrA2 and CtrA3 are thermophilic proteins with an activity maximum at 75 degrees C. Electron cryomicroscopy of two-dimensional crystals of CtrA3 yielded a projection map at approximately 7 A resolution with density peaks, indicating eight membrane-spanning alpha-helices per monomer. A fit of the Ca-ATPase structure to the projection map indicates that the arrangement of the six central helices surrounding the ion-binding site in the membrane is conserved, and suggests the position of the two additional N-terminal transmembrane helices that are characteristic of the heavy metal, eight-helix P(1B)-type ATPases.
Asunto(s)
Adenosina Trifosfatasas/química , Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Bacterias/clasificación , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Clonación Molecular , ATPasas Transportadoras de Cobre , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.
Asunto(s)
Uniones Adherentes/metabolismo , Moléculas de Adhesión Celular/metabolismo , Endotelio Vascular/ultraestructura , Uniones Adherentes/química , Antígenos CD , Cadherinas , Moléculas de Adhesión Celular/química , Microscopía por Crioelectrón , Endotelio Vascular/metabolismo , Humanos , LiposomasRESUMEN
A high concentration of cadherin molecules at cell-cell adhesion sites is believed to be essential for generating strong intercellular junctions. In order to determine the interactions of cadherin domains involved in the early stages of lateral cluster formation on the cell surface, a recombinant fragment encompassing the first four domains of human VE-cadherin with a His-tag at the C terminus (VE-EC1-4-His) was produced. Two-dimensional crystals of VE-EC1-4-His were formed at the air-water interface using conventional lipids modified to contain a Ni(2+)-chelating group, which provides a specific site for interaction with the polyhistidine tag. The VE-EC1-4-His was monomeric at the concentration employed for crystal formation; however, the crystals exhibited a p2 symmetry and the presence of cis-dimer interactions between symmetry-related molecules. The VE-EC1-4-His molecules in the crystalline array have a remarkably compact conformation in contrast to the elongated "string of pearls" conformation seen in the hexameric assembly of VE-EC1-4-His in solution, and as seen in the crystal structure of C-cadherin. These results indicate that VE-cadherin can exist in at least two oligomeric states with different interactions between domains and can adopt highly different conformational states. We suggest that the compact cis-dimeric state may occur on isolated cells and that the compact form may serve to protect the molecule from degradation. As previously proposed we suppose that the trans-hexameric form is involved in intercellular adhesion.