Andrew P. VanDemark

Molecular Insights into Polyubiquitin Chain Assembly: Crystal Structure of the Mms2/Ubc13 Heterodimer

While the signaling properties of ubiquitin depend on the topology of polyubiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyubiquitin chains that act as a novel signal in postreplicative DNA repair and I kappa B alpha kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyubiquitin signals.

Potent D-peptide inhibitors of HIV-1 entry

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D-peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC50 = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS.

Structural and Functional Analysis of the Spt16p N-terminal Domain Reveals Overlapping Roles of yFACT Subunits

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.

Structural basis of ubiquitylation

The attachment of the small protein ubiquitin to other proteins, a process known as ubiquitylation, is a widespread form of post-translational modification that regulates numerous cellular functions in eukaryotes. Ubiquitylation is performed by complexes of E2 and E3 enzymes that are assembled and select substrates via a series of protein-protein interactions. Recent structure determinations of the ubiquitylation machinery have revealed some of the various protein-protein interfaces involved.

Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin

Significance The polymerase associated factor 1 complex (Paf1C) is an RNA polymerase (pol) II accessory factor that broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription. This study shows how phosphorylation of a repeated motif within an additional factor, Spt5, is recognized and used by the Plus3 domain within the Paf1C subunit Rtf1 (restores TBP function 1) to promote recruitment of Paf1C to the transcription machinery. Deletions of both the Rtf1 Plus3 domain and the C domain of Cdc73 (Cell Division Cycle 73) are required to abolish Paf1C-mediated histone modifications and chromatin occupancy suggesting that dual attachment points facilitate the association of Paf1C with RNA pol II. Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.

Opening doors into the proteasome

Two new crystal structures suggest that the eukaryotic proteasome is a gated protease and that opening of the gate may be a regulated event.

A novel Drosophila SOD2 mutant demonstrates a role for mitochondrial ROS in neurodevelopment and disease

Reactive oxygen species (ROS) play essential roles in cell signaling, survival, and homeostasis. Aberrant ROS lead to disease and contribute to the aging process. Numerous enzymes and vigilant antioxidant pathways are required to regulate ROS for normal cellular health. Mitochondria are a major source of ROS, and mechanisms to prevent elevated ROS during oxidative phosphorylation require super oxide dismutase (SOD) activity. SOD2, also known as MnSOD, is targeted to mitochondria and is instrumental in regulating ROS by conversion of superoxides to hydrogen peroxide, which is further broken down into H2O and oxygen. Here, we describe the identification of a novel mutation within the mitochondrial SOD2 enzyme in Drosophila that results in adults with an extremely shortened life span, sensitivity to hyperoxia, and neuropathology. Additional studies demonstrate that this novel mutant, SOD2bewildered, exhibits abnormal brain morphology, suggesting a critical role for this protein in neurodevelopment. We investigated the basis of this neurodevelopmental defect and discovered an increase in aberrant axonal that could underlie the aberrant neurodevelopment and brain morphology defects. This novel allele, SOD2bewildered, provides a unique opportunity to study the effects of increased mitochondrial ROS on neural development, axonal targeting, and neural cell degeneration in vivo.

Cdc73 Subunit of Paf1 Complex Contains C-terminal Ras-like Domain That Promotes Association of Paf1 Complex with Chromatin

Background: The Paf1 complex associates with RNA polymerase II during elongation. Results: The Cdc73 C-domain adopts a Ras-like fold that contributes to histone methylation and Paf1C recruitment to active genes. Conclusion: Cdc73 C-domain is important for Paf1 complex recruitment to genes. Significance: Rtf1 and Cdc73 C-domain combine to couple Paf1 complex to elongating polymerase. The conserved Paf1 complex localizes to the coding regions of genes and facilitates multiple processes during transcription elongation, including the regulation of histone modifications. However, the mechanisms that govern Paf1 complex recruitment to active genes are undefined. Here we describe a previously unrecognized domain within the Cdc73 subunit of the Paf1 complex, the Cdc73 C-domain, and demonstrate its importance for Paf1 complex occupancy on transcribed chromatin. Deletion of the C-domain causes phenotypes associated with elongation defects without an apparent loss of complex integrity. Simultaneous mutation of the C-domain and another subunit of the Paf1 complex, Rtf1, causes enhanced mutant phenotypes and loss of histone H3 lysine 36 trimethylation. The crystal structure of the C-domain reveals unexpected similarity to the Ras family of small GTPases. Instead of a deep nucleotide-binding pocket, the C-domain contains a large but comparatively flat surface of highly conserved residues, devoid of ligand. Deletion of the C-domain results in reduced chromatin association for multiple Paf1 complex subunits. We conclude that the Cdc73 C-domain probably constitutes a protein interaction surface that functions with Rtf1 in coupling the Paf1 complex to the RNA polymerase II elongation machinery.

The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55–Rad57 to mediate error-free recombination

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2–Psy3 heterodimer preferentially binds synthetic forked DNA or 3′-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55–Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55–Rad57. Importantly, Csm2’s interaction with Rad51 is dependent on Rad55, whereas Csm2’s interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2–Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55–Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.

Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.