Eukaryotic transcriptional coactivators are multi-subunit complexes that both modify chromatin and recognize histone modifications. Until recently, structural information on these large complexes has been limited to isolated enzymatic domains or chromatin-binding motifs. This review summarizes recent structural studies of the SAGA coactivator complex that have greatly advanced our understanding of the interplay between its different subunits. The structure of the four-protein SAGA deubiquitinating module has provided a first glimpse of the larger organization of a coactivator complex, and illustrates how interdependent subunits interact with each other to form an active and functional enzyme complex. In addition, structures of the histone binding domains of ATXN7 and Sgf29 shed light on the interactions with chromatin that help recruit the SAGA complex.
Histones are ubiquitinated in response to DNA double strand breaks (DSB), promoting recruitment of repair proteins to chromatin1. UBC13 (UBE2N) is an ubiquitin conjugating enzyme (E2) that heterodimerizes with UEV1a2 and synthesizes K63–linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF1683. K63Ub synthesis is regulated in a noncanonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13~Ub thiolester4. Residues N-terminal to the OTU domain, which had been implicated in ubiquitin binding5, are required for binding to UBC13~Ub and inhibition of K63Ub synthesis5. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find 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, while at the same time disrupting interactions with UEV1a in a manner that depends upon the OTUB1 N-terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that 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 transfer6,7. 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 enzymes4 in a non-catalytic manner.
Ubiquitination is a widely-studied regulatory modification involved in protein degradation, DNA damage repair and the immune response. Conjugation of ubiquitin to a substrate lysine occurs in an enzymatic cascade involving an E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, or E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
Ubiquitin Conjugation; Ubiquitin-like proteins; Ubc13-Mms2; ubiquitin-activating enzyme
Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity and cell proliferation, though few cellular ADP-ribosyltransferases have been identified. The sirtuins are a highly conserved family of protein deacetylases and several family members have also been reported to carry out protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog from Trypanosoma brucei, TbSIR2RP1, on both acetylated and unacetylated substrates. We demonstrate that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction carried out by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and mass spectrometry data show that arginine is the major ADP-ribose acceptor for unacetylated substrates, while arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We carried out combined ab initio QM/MM molecular dynamics simulations, which indicate that sirtuin ADP-ribosylation at arginine is energetically feasible, and employs a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can carry out ADP-ribosylation using two distinct mechanisms that depend upon whether or not the substrate is acetylated.
Pax5 is a critical regulator of transcription and lineage commitment in B lymphocytes. In B cells, mb-1 (Ig-α) promoter transcription is activated by Pax5 through its recruitment of Ets family proteins to a composite site, the P5-EBS (Pax5-Ets binding site). Previously, X-ray crystallographic analysis revealed a network of contacts between the DNA binding domains of Pax5 and Ets-1 while bound to the P5-EBS. Here, we report that Pax5 assembles these ternary complexes via highly cooperative interactions that overcome the autoinhibition of Ets-1. Using recombinant proteins, we calculated KD(app) values for the binding of Pax5, Ets-1 and GABP proteins, separately or together, to the P5-EBS. By itself, Pax5 binds the P5-EBS with high affinity (KD ≅ 2 nM). Ets-1(331–440) bound the P5-EBS by itself with low affinity (KD = 136 nM). However, autoinhibited Ets-1(280–440) alone does not bind detectably to the suboptimal sequences of the P5-EBS. Recruitment of Ets-1(331–440) or (280–440) resulted in highly efficient ternary complex assembly with Pax5. Pax5 counteracts autoinhibition and increases binding of Ets-1 of the mb-1 promoter by >1000-fold. Mutation of Pax5 Gln22 to alanine (Q22A) enhances promoter binding by Pax5; however, Q22A greatly reduces recruitment of Ets-1(331–440) and (280–440) by Pax5 (8.9– or >300–fold, respectively). Thus, Gln22 of Pax5 is essential for overcoming Ets-1 autoinhibition. Pax5 wild type and Q22A each recruited GABPα/β1 to the mb-1 promoter with similar affinities, but recruitment was less efficient than that of Ets-1 (reduced by ~8–fold). Our results suggest a mechanism that allows Pax5 to overcome autoinhibition of Ets-1 DNA binding. In summary, these data illustrate requirements for partnerships between Ets proteins and Pax5.
Pax; Ets; GABP; DNA binding; autoinhibition
Ubiquitination involves the covalent attachment of the ubiquitin C-terminus to the lysine sidechain of a substrate protein by an isopeptide bond. The modification can comprise a single ubiquitin moiety or a chain of ubiquitin molecules joined by isopeptide bonds between the C-terminus of one ubiquitin with one of the seven lysine residues in the next ubiquitin. Modification of substrate proteins with Lys63-linked polyubiquitin plays a key non-degradative signaling role in many biological processes including DNA repair and NF-κB activation, whereas substrates modified by lysine-48 (Lys48) linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked ubiquitin chains presumably stems from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetraubiquitin at a resolution of 1.96 Å and performed Small Angle X-ray scattering (SAXS) experiments and molecular dynamics (MD) simulations to probe the conformation of Lys63 tetraubiquitin in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 Ub4. Small Angle X-ray scattering (SAXS) experiments show that the tetraubiquitin chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 polyubiquitin chains.
Sirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD+-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. We have studied the ADP-ribosylation activity of a bacterial sirtuin, Sir2Tm, and show that acetylated peptides containing arginine or lysine 2 residues C-terminal to the acetyl lysine, the +2 position, are preferentially ADP-ribosylated at the +2 residue. A structure of Sir2Tm bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide. The new biochemical and structural studies presented here provide mechanistic insights into the Sir2 ADP-ribosylation reaction and will aid in identifying substrates of this reaction.
Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.
deubiquitination; isopeptide; linkage specificity; otubain; polyubiquitin
Sirtuin enzymes comprise a unique class of NAD+-dependent protein deacetylases. Although structures of a number of sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD+ analogue and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.
Sir2; Sirtuin; O-alkylamidate; S-alkylamidate; DADMe-NAD+
The yeast Rap1 protein plays an important role in transcriptional silencing and in telomere length homeostasis. Rap1 mediates silencing at the HM loci and at telomeres by recruiting the Sir3 and Sir4 proteins to chromatin via a Rap1 C-terminal domain, which also recruits the telomere length regulators, Rif1 and Rif2. We report the 1.85 Å resolution crystal structure of the Rap1 C-terminus, which adopts an all-helical fold with no structural homologues. The structure was used to engineer surface mutations in Rap1, and the effects of these mutations on silencing and telomere length regulation were assayed in vivo. Our surprising finding was that there was no overlap between mutations affecting mating-type and telomeric silencing, suggesting that Rap1 plays distinct roles in silencing at the silent mating-type loci and telomeres. We also found novel Rap1 phenotypes and new separation-of-function mutants, which provide new tools for studying Rap1 function. Yeast two-hybrid studies were used to determine how specific mutations affect recruitment of Sir3, Rif1, and Rif2. A comparison of the yeast two-hybrid and functional data revealed patterns of protein interactions that correlate with each Rap1 phenotype. We found that Sir3 interactions are important for telomeric silencing, but not mating-type, silencing, and that Rif1 and Rif2 interactions are important in different subsets of telomeric length mutants. Our results show that the role of Rap1 in silencing differs between the HM loci and telomeres and offer insight into the interplay between HM silencing, telomeric silencing, and telomere length regulation. Our findings suggest a model in which competition and multiple recruitment events modulate silencing and telomere length regulation.
Rap1; Sir complex; Rif proteins; transcriptional silencing; telomere length
Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by the administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for β cell function, suggesting a vital framework for the regulation of glucose homeostasis.
Hoogsteen base pairs within duplex DNA typically are only observed in regions containing significant distortion or near sites of drug intercalation. We report here the observation of a Hoogsteen base pair embedded within undistorted, unmodified B-DNA. The Hoogsteen base pair, consisting of a syn adenine base paired with an anti thymine base, is found in the 2.1 Å resolution structure of the MATα2 homeodomain bound to DNA in a region where a specifically and a non-specifically bound homeodomain contact overlapping sites. NMR studies of the free DNA show no evidence of Hoogsteen base pair formation, suggesting that protein binding favors the transition from a Watson–Crick to a Hoogsteen base pair. Molecular dynamics simulations of the homeodomain–DNA complex support a role for the non-specifically bound protein in favoring Hoogsteen base pair formation. The presence of a Hoogsteen base pair in the crystal structure of a protein–DNA complex raises the possibility that Hoogsteen base pairs could occur within duplex DNA and play a hitherto unrecognized role in transcription, replication and other cellular processes.