ATP-Mediated Conformational Changes in the RecA Filament

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.DNA binding protein ͉ genetic transformation ͉ type IV secretion ͉ x-ray crystallography ͉ protein conformation

Section: Discussionmentioning

confidence: 64%

“…RecA is a well known example, where the protein subunits in complex with ssDNA were rotated with respect to those in a crystal structure obtained in the absence of ssDNA (32). In the structure of human replication protein A (RPA), large conformational changes within individual OB folds are induced in the presence of ssDNA as well (39).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners

Dym¹,

Albeck²,

Unger³

et al. 2008

“…2 A) that has been proposed, based on the structure of RecA, to activate the water nucleophile for in-line attack of the ATP ␥-phosphate (23,24). RecA itself forms helical filaments (23,26), but also hexameric ring structures of unknown function (27).…”

Section: Resultsmentioning

confidence: 99%

Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses

Mori

Pattanayek

et al. 2004

134

192

In cyanobacteria, KaiC is an essential hexameric clock protein that forms the core of a circadian protein complex. KaiC can be phosphorylated, and the ratio of phospho-KaiC to non-phospho-KaiC is correlated with circadian period. Structural analyses of KaiC crystals identify three potential phosphorylation sites within a 10-Å radius of the ATP binding regions that are at the T432, S431, and T426 residues in the KaiCII domains. When these residues are mutated by alanine substitution singly or in combination, KaiC phosphorylation is altered, and circadian rhythmicity is abolished. These alanine substitutions do not prevent KaiC from hexamerizing. Intriguingly, the ability of KaiC overexpression to repress its own promoter is also not prevented by alanine substitutions at these sites, implying that the capability of KaiC to repress its promoter is not sufficient to allow the clockwork to oscillate. The KaiC structure and the mutational analysis suggest that S431 and T426 may share a phosphate that can shuttle between these two residues. Because the phosphorylation status of KaiC oscillates over the daily cycle, and KaiC phosphorylation is essential for clock function as shown here, daily modulations of KaiC activity by phosphorylation at T432 and S431͞T426 seem to be key components of the circadian clockwork in cyanobacteria.

“…Nevertheless, it is likely that this short motif is part of the subunit-subunit interface in both RAD51 and RecA filaments, and the binding of the BRC repeats to this site blocks filament formation. In fact, a portion of this motif was part of the subunit-subunit interface in an EM-based model for the active RecA-DNA filament (43). This model for a RecA-DNA-ATP filament differed from the filament seen in a crystal of RecA alone (in the absence of both DNA and ATP) by a rotation of subunits through Ϸ30°.…”

Section: Comparison With a Previous Interpretationmentioning

confidence: 99%

BRCA2 BRC motifs bind RAD51–DNA filaments

Galkin

Esashi

Xiong

et al. 2005

Self Cite

127

118

Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.electron microscopy ͉ nucleoprotein filaments ͉ recombination ͉ genome stability and dynamics ͉ structural biology M utations in BRCA2 are associated with an increased risk of breast and ovarian cancers, and almost half the cases of inherited early onset breast cancers have been linked to mutations in BRCA2. The product of this gene, BRCA2 protein (384 kDa), interacts with RAD51, and it has been shown that both proteins are essential for homologous recombination, DNA repair, and the maintenance of genome stability. There appear to be a number of different interactions between BRCA2 and RAD51. One such interaction involves a C-terminal region of BRCA2 (1). In addition, BRCA2 interacts with RAD51 through the eight conserved BRC repeats in BRCA2 (2, 3), and mutations within these repeats are associated with cancer predisposition. The deletion of several BRC repeats in mice leads to cancer (4), and somatic mutations in BRC repeats have been found to be associated with breast cancer (5). Although there are eight BRC repeats in human BRCA2, in other organisms the number of BRC repeats is quite variable, with 15 found in a Trypanosoma BRCA2-like protein (6) and only one found in the Ustilago Brh2 protein (7).Many details of the interactions between BRCA2 and RAD51 are unclear, such as whether BRCA2 binds to the N-terminal domain of RAD51 (8) or to the nucleotide-binding core (3, 9). Recently, a fusion protein containing the nucleotide-binding core of RAD51 and a single BRC repeat was crystallized. Analysis of the x-ray structure of the fusion led t...