Activators of bacterial σ 54 -RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF , bound to an ATP transition state analog (ADP.AlF x ), in complex with its basal factor σ 54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF at 1.75 Å into the EM map we identify two loops involved in binding σ 54 . By comparing enhancerbinding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ 54 .Gene expression is regulated at the level of RNA polymerase (RNAP) activity. Bacterial RNAP containing the σ 54 factor requires specialized activator proteins, referred to as bacterial Enhancer-Binding Proteins (EBPs) that interact with the basal transcription complex from remote DNA sites by DNA looping (1-4). EBPs bind Upstream Activating Sequences (UAS) via their C-terminal DNA-binding domains and form higher order oligomers that use ATP-hydrolysis to activate transcription (5, 6). EBPs' central σ 54 -RNAP interacting domain is responsible for ATPase activity and transcription activation (7-9) and belongs to the larger AAA+ (ATPase Associated with various cellular Activities) family of proteins (10-12). Well studied EBPs include Phage Shock protein F (PspF), nitrogen fixation protein A (NifA), nitrogen regulation protein C (NtrC), and C 4 -dicarboxylic acid transport protein D (DctD) (1-3, 7, 13).PspF from Escherichia coli forms a stable oligomeric complex with σ 54 at the point of ATP hydrolysis (14). PspF-ADP.AlF x alters the interaction between σ 54 and promoter DNA similarly to PspF hydrolyzing ATP (15), and was thus deemed a functional hydrolysis intermediate. Activator nucleotide-hydrolysis dependent events couple the chemical energy of hydrolysis to transcriptional activation. The highly conserved and EBP-specific GAFTGA amino acid motif (Fig. S1) is a crucial mechanical determinant for the successful transfer of energy from ATP hydrolysis in EBP to the RNAP holoenzyme via σ 54 's small N-terminal * To whom correspondence should be addressed. xiaodong.zhang@imperial.ac.uk. The lack of structural information has hindered progress towards understanding the basis of this energy transfer process required for transcriptional activation. We now present a structure-function analysis of one such system using: 1) a cryo-electron microscopy reconstruction of PspF's AAA+ domain (residues 1-275, PspF ) in complex with σ 54 at the point of ATP hydrolysis (mimicked by in-situ formed ADP.AlF x ), 2) the crystal structure of apo PspF (1-275) at 1.75 Å resolution, and 3) mutational analysis. Europe PMC Funders GroupNano-electro spray mass spectroscopy of a PspF (1-275) -σ 54 complex with ADP.AlF x established that six monomers of PspF are in complex with a monomeric σ 54 , consistent with AAA+ proteins functioning as hexamers (10, 12).The 3-dimensional reconstruction of the...

Background: p97/VCP disease-linked mutations increase ATPase activity and destabilize the N-D1 domain interaction.Results: Increased N-domain flexibility in p97/VCP increases ATPase activity, whereas locking down the N-domain decreases it.Conclusion: The p97/VCP N-domain position relative to the D1 ring is linked to ATP hydrolysis ability.Significance: p97/VCP N-domain conformational changes cause transitions between an active and inactive state.

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An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

To understand the role of the type 2A-like protein phosphatase in the cell division cycle, we investigated the mutant phenotypes obtained when the fission yeast ppal § and ppa2 § phosphatase genes (which encode polypeptides with -80% identity to mammalian type 2A phosphatases) were either deleted or overexpressed. We also investigated the in vivo effect of okadaic acid, an inhibitor of protein serine/threonine phosphatases, on cell division. We show that ppa2 + interacts genetically with the cell cycle regulators cdc25 § and wee1 § as a ppa2 deletion is lethal when combined with wee1-50 but partially suppresses the conditional lethality of cdc25-22 mutation. Evidence that ppa2 § negatively controls the entry into mitosis, possibly through the regulation of cdc2 tyrosine phosphorylation, is presented, ppa2 phosphatase is abundant in the cytoplasm, in contrast to the type l-like phosphatase dis2, which is enriched in the nucleus. Overproduced ppal or ppa2 proteins accumulate in the cytoplasm near the nuclear periphery, and cells arrest in interphase. Okadaic acid-treated cells, like a ppa2 deletion, are short in length and display protein hyperphosphorylation. Cytokinesis is also inhibited, producing binucleated cells. We show that ppa2 is the genetic locus controlling okadaic acid sensitivity. The ppa2 deletion reveals the same hyperphosphorylated proteins as okadaic acid. When a strain deleted for ppa2 is treated with okadaic acid, cell size is reduced further to that of weel-50 mutant strain or overexpressing the cdc25 + gene product, suggesting functional relationship of ppa2 with the cdc25 tyrosine phosphatase and/or the wee1 kinase in cell cycle control. (Ohkura et al. 1989) genes, which encode polypeptides highly similar (-80% identical) to PP1. A Drosophila mutant in one of the four PPl-related genes was found to produce a similar mitotic defect (Axton et al. 1990). Definitive evidence for the essentiality of PP1-related phosphatases in cell division was provided in fission yeast by the lethality of the double gene disruption of dis2 + and sds21 + (designated hdis2-Asds21); the latter gene codes for a polypeptide highly similar (-79%) to dis2 + (Ohkura et al, 1989). Single gene deletion mutants (Adis2 or dtsds21) were viable.Other phosphatase genes encoding polypeptides highly similar (-80% identical) to PP2A are present in fission and budding yeasts (Kinoshita et al. 1990;Sneddon et al. 1990;Ronne et al. 1991;Sutton et al. 1991). From multiple gene disruption and point mutant analyses, the fission yeast PP2A-related phosphatase genes ppal + and 1Present address:

The AAA (ATPase associated with various cellular activities) p97 [also known as VCP (valosin-containing protein)] participates in numerous biological activities and is an essential component of the ubiquitin signalling pathway. A plethora of adaptors have been reported for p97, and increasing evidence is suggesting that it is through adaptor binding that p97 is diverted into different cellular pathways. Studying the interaction between p97 and its adaptors is therefore crucial to our understanding of the physiological roles of the protein. The interactions between p97 and the PUB [PNGase (peptide N-glycosidase)/ubiquitin-associated] domain of PNGase, the UBX (ubiquitin regulatory X) domain of p47, and the UBD (ubiquitin D) domain of Npl4 have been structurally characterized. UBX and UBD are structural homologues that share similar p97-binding modes; it is plausible that other proteins that contain a UBX/UBX-like domain also interact with p97 via similar mechanisms. In addition, several short p97-interacting motifs, such as VBM (VCP-binding motif), VIM (VCP-interacting motif) and SHP, have been identified recently and are also shared between p97 adaptors, hinting that proteins possessing the same p97-binding motif might also share common p97-binding mechanisms. In this review, we aim to summarize our current knowledge on adaptor binding to p97.

Backggvound: Protein phosphatase 2A (PP2A) holoenzymes have a trimeric structure, consisting of a catalytic subunit C and two regulatory subunits A (PR65) and B (PR55). In fission yeast the C subunits, being 80% identical to their mammalian counterparts, are essential for viability and negatively regulate the entry into mitosis. Genetic analyses in budding yeast and Drosophila show that the regulatory subunits are implicated in chromosome segregation, cell morphogenesis and/or cytokinesis. Results:We isolated fission yeast genes p a a l + and p a b l + encoding the regulatory subunits PR65 and PR55, respectively. Gene disruption showed that the paa1'-gene was essential for viability while p a b l + was not required at 26-33°C. Microtubule

FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore.