A genome encodes the information needed to produce all of the proteins and nucleic acids that carry out a cell's essential functions. The cell utilizes the information by first transcribing an RNA copy of each gene. The information in the RNA is then translated by the ribosome to produce a protein; for some genes, the RNA itself is the end product. Since only a fraction of the encoded protein and RNA molecules is needed by the cell at any given time, all organisms have elaborate mechanisms for controlling whether a gene is transcribed. Our research is aimed at understanding at a molecular level how eukaryotic cells control gene expression. We use x-ray crystallography to determine the atomic structures of the protein and nucleic acid complexes that participate in gene regulation. Together with biochemical and genetic studies, these structures provide mechanistic insights into the fundamental processes that govern transcription regulation. Our research is also focused on the assembly of Lys63-linked polyubiquitin. These chains of ubiquitin proteins are attached to a variety of cellular targets, where they play important roles in transcriptional activation in the inflammatory response, as well as in the repair of DNA damage. Here, too, the tools of structural biology are revealing how the cell assembles these chains with the correct linkages and attaches them to other protein complexes in the cell.

Transcriptional Silencing
The DNA in eukaryotic cells is packaged by histone proteins, giving rise to a nucleoprotein complex called chromatin. Some regions of chromatin are relatively accessible to the transcriptional machinery and contain actively transcribed genes; other regions are silenced and are organized in a way that prevents transcription. Transcriptional silencing is brought about by protein complexes that chemically modify histone proteins, either by introducing post-translational modifications or by removing them. We study the enzymes and protein complexes that establish and maintain transcriptional silencing. One example is Sir2, an enzyme that deacetylates 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, and enzyme regulation.

Sir2 proteins play an intriguing role in life span, with high levels of Sir2 activity extending life span and low levels of Sir2 activity leading to shortening of life span in yeast, worms, and flies. Members of the Sir2 enzyme family also regulate important pathways in humans that are involved in protection of neurons from degeneration, fat mobilization, and cell death. Sir2 activity is linked to cellular concentrations of NAD+, a substrate of Sir2, as well as to levels of nicotinamide, a reaction product that inhibits Sir2 activity. Some Sir2 enzymes catalyze a somewhat different reaction in which NAD+ is cleaved to produce ADP ribose, which is then attached to a substrate in a reaction known as mono-ADP ribosylation. Understanding the chemistry and regulation of Sir2 proteins, as well as the basis for the dual enzymatic activity, is central to unraveling how these enzymes help to regulate different cellular pathways.

Our structural studies of Sir2 enzymes bound to a variety of substrates, products, and inhibitors have revealed how these proteins carry out their unique chemical reaction in the cell. The structure of Sir2Af2 bound to an acetylated peptide corresponding to the carboxyl terminus of p53 showed that peptides bind in a cleft between the two domains of the enzyme, closing the intervening gap and inserting the acetyl-lysine side chain deep within the enzyme active site. Structural and biochemical studies of a bacterial sirtuin, Sir2Tm, bound to a variety of peptide substrates have shown that interactions between the enzyme and the substrate residues flanking the acetyl-lysine influence the peptide substrate specificity of the enzyme. We have also determined structures of Sir2 enzymes bound simultaneously to both peptide and NAD+ before catalysis, as well as after the enzymatic reaction has occurred in the crystal.

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. They have shown us that acetyl-lysine induces binding of NAD+ in a strained conformation that favors cleavage of the nicotinamide ring from NAD+. This cleavage sets up a cascade of events leading to the eventual deacetylation of lysine and the release of the reaction products. To determine structures of a series of trapped reaction intermediates, we will address how subsequent steps in the reaction are catalyzed by the enzyme. These studies may also explain why some members of the Sir2 family catalyze the transfer of the ADP-ribose portion of NAD+ to a protein substrate, a reaction that is known as ADP ribosylation.

The activity of Sir2 enzymes can be controlled by small molecules that bind within the enzyme active site. We have determined how nicotinamide, a product of the NAD+-dependent deacetylation reaction, binds in the enzyme active site and interferes with the deacetylation reaction by promoting a back reaction known as base exchange. We have shown that Sir2 enzymes contain a single site that is involved in both NAD+ binding and nicotinamide inhibition. We are studying how other small molecules either repress or stimulate the enzymatic activity of Sir2 enzymes.

In some organisms, the transcription of genes that lie near the ends of chromosomes—known as telomeres—is subject to silencing mediated by Sir2 enzymes. This type of silencing is found in budding yeast, as well as in a pathogenic yeast, Candida glabrata, and in the malaria parasite. This transcriptional repression, known as telomeric silencing, requires additional proteins that help recruit Sir2 enzymes. Structural studies of the telomere-binding protein Rap1 and of the silencing proteins recruited by Rap1 are shedding light on how telomeric silencing is established and maintained.

The nucleus contains a set of enzymes that synthesize NAD+ from a variety of molecular precursors. The relative activity of these enzymes controls Sir2 activity by affecting the nuclear concentrations of NAD+ and nicotinamide. We have determined the structure of a mammalian NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (Nampt), bound to reaction products. Nampt catalyzes the synthesis of nicotinamide mononucleotide from nicotinamide and phosphoribosylpyrophosphate in a reaction that is enhanced when the enzyme becomes autophosphorylated. Ongoing structural studies, as well as biochemical studies of mutant and wild-type enzymes, are being carried out to elucidate the reaction mechanism and determine the role that autophosphorylation plays in the catalytic mechanism.

Assembly of Lys63-Linked Polyubiquitin Chains
Ubiquitin is a small protein that serves as the building block for chains of polyubiquitin, which are attached to other proteins and thereby mediate a variety of biological processes. Polyubiquitin is formed when the C terminus of one ubiquitin is joined to one of the seven surface lysines of ubiquitin. Polyubiquitin chains with different types of lysine linkages play distinct biological roles. For example, Lys48-linked polyubiquitin chains target proteins for destruction by the proteasome, whereas Lys63-linked polyubiquitin chains are nondegradative signals that play a role in DNA damage tolerance and NF?B activation.

We study the molecular basis for Lys63-linked polyubiquitin chain assembly. 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 Mms2/Ubc13, as well as a complex containing ubiquitin covalently joined to the active-site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Ub~Ubc13/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. We continue to study polyubiquitin chain assembly by Mms2/Ubc13, as well as how it cooperates with the ubiquitin ligase, Rad5, to modify substrates with Lys63-linked polyubiquitin chains.

A portion of this work has been supported by the National Science Foundation and the National Institutes of Health.

Last updated: May 2, 2007.