Protein function is dynamically regulated in the cell by the attachment and subsequent removal of covalent posttranslational modifications. The modifications can be small, as in the case of acetylation, or quite large, as in the case of a polyubiquitin chain. Acetylation and ubiquitination both occur in chromatin, the nucleoprotein complex into which eukaryotic DNA is packaged. Acetylation of the histone proteins in chromatin is associated with activation of transcription, whereas ubiquitination can be either an activating or a repressive mark, depending on which histone protein is modified. Ubiquitination of chromatin also plays a role in the response to DNA double-strand breaks, helping to recruit proteins that are required for DNA repair. We are interested in the molecular basis for these events, which ensure the integrity and expression of the genome. We use x-ray crystallography, enzymology, and a variety of biophysical tools to gain insights into the mechanisms underlying these essential cellular processes.
Ubiquitin Signaling
Ubiquitin is a small protein that regulates a wide array of cellular processes through its covalent conjugation to cellular proteins. In addition to its well-characterized role in targeting proteins for proteasomal degradation, there are now numerous examples of nondegradative roles for ubiquitin modification. Ubiquitin is attached to substrate lysines through a cascade of enzymatic reactions catalyzed by the E1, E2, and E3 enzymes. Downstream factors recognize the ubiquitin modification, which can consist of a single ubiquitin or a polyubiquitin chain composed of ubiquitin monomers joined by covalent bonds between the C terminus of one ubiquitin and one of the seven lysine side chains in the next ubiquitin. Polyubiquitin chains with different types of lysine linkages play distinct biological roles, as does monoubiquitin.
Assembly of K63-Linked Polyubiquitin in the DNA Damage Response
Lys63-linked polyubiquitin chains are nondegradative signals that play a role in DNA damage tolerance and the inflammatory response. Lys63-linked chains are assembled by a specialized heterodimer that helps to catalyze formation of a covalent bond between Lys63 of one ubiquitin and the C terminus of the next. We determined the structure of the yeast enzyme Ubc13/Mms2 as well as that of the reaction intermediate in which the C terminus of ubiquitin is covalently joined to the active-site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Ubc13~Ub/Mms2 complex to the acceptor-binding site of Mms2/Ubc13 in an adjacent complex allowed us to visualize the molecular determinants of acceptor-ubiquitin binding and explain how Mms2 helps to orient ubiquitin to give rise to a Lys63-linked polyubiquitin chain. Our ongoing studies are focused on how Ubc13/Mms2 cooperates with E3 ligases in both yeast and human cells to ubiquitinate PCNA and histones in response to DNA damage.
Non-Catalytic Regulation of K63 Polyubiquitination by the Deubiquitinating Enzyme, OTUB1
Histones are ubiquitinated in response to DNA double-strand breaks, promoting recruitment of repair proteins to chromatin. The human UBC13/UEV1A heterodimer synthesizes K63-linked polyubiquitin chains (K63Ub) at double-strand break sites in concert with the ubiquitin E3 ligase, RNF168. The deubiquitinating enzyme, OTUB1, regulates K63Ub synthesis non-catalytically by binding to the UBC13~Ub thiolester and preventing ubiquitin conjugation. The flexible N-terminus of OTUB1, which we had previously found to contain a ubiquitin-binding region, is required for both inhibition of K63Ub synthesis and binding of OTUB1 to UBC13~Ub. Using a combination of structural and biochemical studies, we determined the mechanism by which OTUB1 inhibits ubiquitination. We unexpectedly found that OTUB1 binding to UBC13~Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13~Ub. We also found that OTUB1 disrupts binding to UBC13 of UEV1a, which is required for polyubiquitin synthesis. The structure of OTUB1 bound to an allosteric ubiquitin monomer as well as to a UBC13~Ub conjugation showed how binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.
Monoubiquitination and the Regulation of Transcription
Eukaryotic genes are activated by macromolecular complexes that catalyze the addition and removal of posttranslational histone modifications, facilitating transcription through chromatin. The yeast SAGA complex has been a paradigm for understanding the connection between histone modifications and gene activation as well as the cross-talk between different types of modifications. SAGA functions in a cascade of posttranslational modifications that begins with monoubiquitination of histone H2B-Lys123 by the E2/E3 enzyme pair Rad6/Bre1. This triggers recruitment of a methyltransferase that methylates histone H3 at Lys4, which in turn triggers recruitment of the SAGA complex. SAGA removes monoubiquitin from histone H2B and acetylates histone H3. The transient ubiquitination of H2B is thought to be important for evicting nucleosomes from the transcribed region during transcription initiation and elongation. SAGA consists of 19 proteins that are widely conserved from yeast to humans and are organized into submodules with distinct functions.
We have been studying the SAGA deubiquitinating module (DUBm), a subcomplex of four proteins: the Ubp8 ubiquitin hydrolase and Sus1, Sgf11, and Sgf73, all of which are required for deubiquitination activity. We determined the structure of the DUB module with and without ubiquitin aldehyde, which revealed an unexpected intertwined arrangement of proteins. The structure suggests how specific interactions with both Sgf11 and Sgf73 may position the Ubp8 active-site residues and ubiquitin-binding pocket, thus making Ubp8 active. We are testing these hypotheses with further structural and biophysical studies. We are also studying how the SAGA DUBm is targeted to monoubiquitinated histone H2B.
The Sir2 Family of NAD+-Dependent Deacetylases
The Sir2 enzyme, which was first identified for its role in transcriptional silencing in yeast, is the founding member of an unusual class of enzymes that deacetylate lysine side chains in an unusual reaction that requires NAD+. Enzymes similar to Sir2 are found in virtually all organisms, where they are involved in a variety of critical biological processes, including transcriptional silencing, DNA repair, chromosome stability, neuronal degeneration and fat mobilization. In addition to their principal activity in deacetylating substrates, can also remove other types of acyl modification from residues such as propionyl- and butyryl-lysine while others can mono ADP risobylate substrates. Understanding the novel chemistry of these enzymes and how it is exploited to regulate sirtuin activity in the cell has been an ongoing focus of our research.
We have been investigating the chemical reactions catalyzed by sirtuins by determining structures of these enzymes bound to a variety of substrates and products as well as in complex with reaction intermediates. Each of these structures has provided a snapshot of the reaction, beginning with the binding of sirtuins to acetylated peptide and NAD+, through the release of the products nicotinamide, O-acetyl ADP-ribose, and the deacetylated peptide. We have also determined the structure of a trapped covalent reaction intermediate bound to a sirtuin, which we were able to do by using a thioacetylated peptide. The structure of this complex revealed how side chains within the active site promote the initial cleavage of NAD+ and rearrange to promote subsequent steps in the reaction. These structures have shed light on the unique mechanism by which Sir2 enzymes catalyze the cleavage of NAD+ and the transfer of an acetyl group from the peptide lysine to ADP-ribose as well as how these enzymes are regulated by the metabolite nicotinamide. Mechanistic studies have also shed light on a by-product of sirtuin chemistry: the transfer of the ADP-ribose portion of NAD+ to a protein substrate, a reaction that is known as ADP ribosylation. Using mass spectrometry and protein microarrays in addition to x-ray crystallography, we have pinpointed the determinants for ADP ribosylation and identified the modified side chains.
Portions of this work have been supported by the National Institutes of Health and the National Science Foundation.
As of May 30, 2012
