Structure and regulation of the human INO80-nucleosome complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex.

We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.

The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins.

The cytosolic chaperonin CCT is a 1-MDa protein-folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell-cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8-membered rings stacked back-to-back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co-evolving 'signature residues' that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP-binding heterogeneity and subunit-subunit interactions. The location of the evolutionarily conserved N-terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins.

Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex.

Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.

The interaction network of the chaperonin CCT.

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT-septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.

Structure and dynamics of the yeast SWR1-nucleosome complex.

The yeast SWR1 complex exchanges histone H2A in nucleosomes with Htz1 (H2A.Z in humans). The cryo-electron microscopy structure of the SWR1 complex bound to a nucleosome at 3.6-angstrom resolution reveals details of the intricate interactions between components of the SWR1 complex and its nucleosome substrate. Interactions between the Swr1 motor domains and the DNA wrap at superhelical location 2 distort the DNA, causing a bulge with concomitant translocation of the DNA by one base pair, coupled to conformational changes of the histone core. Furthermore, partial unwrapping of the DNA from the histone core takes place upon binding of nucleosomes to SWR1 complex. The unwrapping, as monitored by single-molecule data, is stabilized and has its dynamics altered by adenosine triphosphate binding but does not require hydrolysis.

Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit.

The eukaryotic cytosolic chaperonin CCT is an essential ATP-dependent protein folding machine whose action is required for folding the cytoskeletal proteins actin and tubulin, and a small number of other substrates, including members of the WD40-propellor repeat-containing protein family. An efficient purification protocol for CCT from Saccharomyces cerevisiae has been developed. It uses the calmodulin binding peptide as an affinity tag in an internal loop in the apical domain of the CCT3 subunit, which is predicted to be located on the outside of the double-ring assembly. This purified yeast CCT was used for a novel quantitative actin-folding assay with human beta-actin or yeast ACT1p protein folding intermediates, Ac(I), pre-synthesised in an Escherichia coli translation system. The formation of native actin follows approximately a first-order reaction with a rate constant of about 0.03 min(-1). Yeast CCT catalyses the folding of yeast ACT1p and human beta-actin with nearly identical rate constants and yields. The results from this controlled CCT-actin folding assay are consistent with a model where CCT and Ac(I) are in a binding pre-equilibrium with a rate-limiting binding step, followed by a faster ATP-driven processing to native actin. In this pure in vitro system, the human beta-actin mutants, D244S and G150P, show impaired folding behaviour in the manner predicted by our sequence-specific recognition model for CCT-actin interaction.

Crosstalk within a functional INO80 complex dimer regulates nucleosome sliding.

Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.

Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cellular and gene expression phenotypes.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

Yeast phosducin-like protein 2 acts as a stimulatory co-factor for the folding of actin by the chaperonin CCT via a ternary complex.

The eukaryotic chaperonin-containing TCP-1 (CCT) folds the cytoskeletal protein actin. The folding mechanism of this 16-subunit, 1-MDa machine is poorly characterised due to the absence of quantitative in vitro assays. We identified phosducin-like protein 2, Plp2p (=PLP2), as an ATP-elutable binding partner of yeast CCT while establishing the CCT interactome. In a novel in vitro CCT-ACT1 folding assay that is functional under physiological conditions, PLP2 is a stimulatory co-factor. In a single ATP-driven cycle, PLP2-CCT-ACT1 complexes yield 30-fold more native actin than CCT-ACT1 complexes. PLP2 interacts directly with ACT1 through the C-terminus of its thioredoxin fold and the CCT-binding subdomain 4 of actin. The in vitro CCT-ACT1-PLP2 folding cycle of the preassembled complex takes 90 s at 30 degrees C, several times slower than the canonical chaperonin GroEL. The specific interactions between PLP2, CCT and ACT1 in the yeast-component in vitro system and the pronounced stimulatory effect of PLP2 on actin folding are consistent with in vivo genetic approaches demonstrating an essential and positive role for PLP2 in cellular processes involving actin in Saccharomyces cerevisiae. In mammalian systems, however, several members of the PLP family, including human PDCL3, the orthologue of PLP2, have been shown to be inhibitory toward CCT-mediated folding of actin in vivo and in vitro. Here, using a rabbit-reticulocyte-derived in vitro translation system, we found that inhibition of beta-actin folding by PDCL3 can be relieved by exchanging its acidic C-terminal extension for that of PLP2. It seems that additional levels of regulatory control of CCT activity by this PLP have emerged in higher eukaryotes.

ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth.

Saccharomyces cerevisiae yeast cells containing the chaperonin CCT (chaperonin-containing t-complex polypeptide 1 (TCP-1)) with the G345D mutation in subunit CCT4 (anc2-1) are temperature-sensitive for growth and display defects in organization of actin structure, budding and cell shape. In this first structure-function analysis of CCT, we show that this mutation abolishes both intra- and inter-ring cooperativity in ATP binding by CCT. The finding that a single mutation in only one subunit in each CCT ring has such drastic effects highlights the importance of allostery for its in vivo function. These results, together with other kinetic data for wild-type CCT reported in this study, provide support for the sequential model for ATP-dependent allosteric transitions in CCT.

Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition.

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.