Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1.

The C-terminal BRCT region of BRCA1 is essential for its DNA repair, transcriptional regulation and tumor suppressor functions. Here we determine the crystal structure of the BRCT domain of human BRCA1 at 2.5 A resolution. The domain contains two BRCT repeats that adopt similar structures and are packed together in a head-to-tail arrangement. Cancer-causing missense mutations occur at the interface between the two repeats and destabilize the structure. The manner by which the two BRCT repeats interact in BRCA1 may represent a general mode of interaction between homologous domains within proteins that interact to regulate the cellular response to DNA damage. The structure provides a basis to predict the structural consequences of uncharacterized BRCA1 mutations.

BRCA1 BRCT Crystal Structure. Scott Williams and Mark Glover.
Williams, R. S., Green, R. and Glover, J. N. M. (2001) Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nature Structural Biology 8(10):838-842. PDF
Crystal structure of the bacterial conjugation repressor FinO.

The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition. Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the surface of the FinO core may present another RNA binding surface.

Crystal structure of the bacterial conjugation repressor FinO. Alex Ghetu and Mark Glover.
Ghetu, A. F., Gubbins, M. J., Frost, L. S. and Glover, J. N. M. (2000) Crystal structure of the bacterial conjugation repressor FinO. Nature Structural Biology 7:565-569. PDF
Structure of the sporulation-specific transcription factor Ndt80 bound to DNA

Progression through the middle phase of sporulation in Saccharomyces cerevisiae is promoted by the successful completion of recombination at the end of prophase I. Completion of meiotic recombination allows the activation of the sporulation-specific transcription factor Ndt80, which binds to a specific DNA sequence, the middle sporulation element (MSE), and activates approximately 150 genes to enable progression through meiosis. Here, we isolate the DNA-binding domain of Ndt80 and determine its crystal structure both free and in complex with an MSE-containing DNA.

Crystal Structure of Ndt80 bound to DNA. Jason Lamoureux and Mark Glover.
Lamoureux, J. S., Stuart, D., Tsang, R., Wu, C. and Glover, J. N. M. (2002) Structure of the sporulation-specific transcription factor Ndt80 bound to DNA. The EMBO Journal 21:5721-5732. PDF
The molecular architecture of the Mammalian DNA repair enzyme, polynucleotide kinase

The Mammalian polynucleotide kinase (PNK) is a bifunctional enzyme, exhibiting 5'-DNA kinase and 3'-phosphatase activities. PNK processes DNA ends at strand breaks to give the ligatable 5'-phosphate and 3'-hydroxyl structures. PNK is involved in the BER and NHEJ pathways, where it participates in the repair of the repair of DNA single- and double-strand breaks as well as in repair of base lesions (BER). PNK is targeted to the repair pathways by the interaction of its N-terminal FHA domain with phosphorylated XRCC1 or XRCC4, key components of BER and NHEJ, respectively.

Mammalian Polynucleotide Kinase PNK Crystal Structure. Nina Bernstein and Mark Glover.
Bernstein, N. K., Williams, R. S., Rakovszky, M. L., Cui, D., Green, R., Karimi-Busheri, F., Mani, R. S., Galicia, S. Koch, C. A., Cass, C. E., Durocher, D., Weinfeld, M. and Glover, J. N. M. (2005) The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase. Molecular Cell 17(5):657-670. PDF
Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Trevor Moraes and Mark Glover.
Moraes, T. F., Edwards, R. A., McKenna, S., Pastushok, L., Xiao, W., Glover, J. N. M. and Ellison, M. J. (2001) Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nature Structure Biology 8:669-673. PDF
Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

The crystal structure of F conjugation regulatory protein, TraM. Jun Lu and Mark Glover.
Lu, J., Edwards, R. A., Wong, J. J. W., Manchak, J. Scott, P. G., Frost, L. S. and Glover, J. N. M. (2006) Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM. EMBO Journal 25(12):2930-2939. PDF
Crystal structure of plasmid pTiC58 VirC2

Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.

Crystal structure of plasmid pTiC58 VirC2. Jun Lu and Mark Glover.
Lu, J., den Dulk-Ras, A., Hooykaas, P. J. and Glover, J. N. M. (2009) Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold. PNAS 106(24):9643-9648. PDF
Crystal Structure Of The Brct Repeat Region From The Mediator of DNA damage checkpoint protein 1, MDC1

MDC1 (mediator of DNA damage checkpoint protein 1) regulates the recognition and repair of DNA double strand breaks in mammalian cells through its interactions with nuclear foci containing the COOH-terminally phosphorylated form of the histone variant, H2AX. Here we demonstrate that the tandem BRCT repeats of MDC1 directly bind to the phosphorylated tail of H2AX-Ser(P)-Gln-Glu-Tyr, in a manner that is critically dependent on the free carboxylate group of the COOH-terminal Tyr residue. We have determined the x-ray crystal structure of the MDC1 BRCT repeats at 1.45 Angstroms resolution. By a comparison with the structure of the BRCA1 BRCT bound to a phosphopeptide, we suggest that two arginine residues in MDC1, Arg(1932) and Arg(1933) may recognize the COOH terminus of the peptide as well as the penultimate Glu of H2AX, while Gln(2013) may provide additional specificity for the COOH-terminal Tyr.

Crystal Structure Of The Brct Repeat Region From The Mediator of DNA damage checkpoint protein 1, MDC1. Megan Lee and Mark Glover.
Lee, M. S.,, Edwards, R. A., Thede, G. L. and Glover, J. N. M. (2005) Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the gamma-H2AX histone tail. JBC 280(37):32053-32056. PDF