Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3–CEN DNA complex by atomic force microscopy. Assembly of CBF3–CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is ≈9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent ≈55° at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.