ATPase Ring Research Articles

Large ring ATPases that organize mitotic chromosomes

Large ring ATPases that organize mitotic chromosomes

Heterohexameric Ring Arrangement of the Eukaryotic Proteasomal ATPases: Implications for Proteasome Structure and Assembly

The proteasome has a paramount role in eukaryotic cell regulation. It consists of a proteolytic core particle (CP) bound to one or two regulatory particles (RPs). Each RP is believed to include six different AAA+ ATPases in a heterohexameric ring that binds the CP while unfolding and translocating substrates into the core. No atomic-resolution RP structures are available. Guided by crystal structures of related homohexameric prokaryotic ATPases, we use disulfide engineering to show that the eukaryotic ATPases form a ring with the arrangement Rpt1-Rpt2-Rpt6-Rpt3-Rpt4-Rpt5 in fully assembled proteasomes. The arrangement is consistent with known assembly intermediates. This quaternary organization clarifies the functional overlap of specific RP assembly chaperones and led us to identify a potential RP assembly intermediate that includes four ATPases (Rpt6-Rpt3-Rpt4-Rpt5) and their cognate chaperones (Rpn14, Nas6, and Nas2). Finally, the ATPase ring structure casts light on alternative RP structural models and the mechanism of RP action.

Crystal Structure of Yeast Rpn14, a Chaperone of the 19 S Regulatory Particle of the Proteasome

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Identification of Caspase-6-Mediated Processing of the Valosin Containing Protein (p97) in Alzheimer's Disease: A Novel Link to Dysfunction in Ubiquitin Proteasome System-Mediated Protein Degradation

The valosin-containing protein (p97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. p97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients' brains, the targeting of p97 by Casp6 may represent an important step that leads to UPS impairment in AD. Here, we show that p97 is a Casp6 substrate in vitro and in vivo. Casp6 cleavage of recombinant p97 generated two N-terminal fragments of 28 and 20 kDa, which were not generated by the other two effector caspases, Caspase-3 and Caspase-7. ATP binding to the D1 ATPase ring of p97 reduced the susceptibility of the N-domain to caspase-mediated proteolysis. Mass spectrometric analysis identified VAPD(179) as a Casp6 cleavage site within p97's N-domain. An anti-neoepitope serum immunohistochemically detected p97 cleaved at VAPD(179) in the cytoplasm of the cell soma and neurites of hippocampal neurons in MCI and AD. Overexpression of p97 (1-179) fragment, representing p97 cleaved at D179, impaired the degradation of model substrates in the ubiquitin-fusion degradation and the N-end rule pathways, and destabilized endogenous p97. Collectively, these results show that p97 is cleaved by Casp6 in AD and suggest p97 cleavage as an important mechanism for UPS impairment.

Functional Consequences of Nucleotide Binding to the Proteasomal ATPases

Protein degradation by the eukaryotic 26S proteasome or the homologous archaeal PAN‐20S proteasome complex is a multistep process that requires ATP hydrolysis by the proteasome‐associated AAA ATPase complex. However, the mechanisms by which these hexameric ATPases utilize ATP to promote protein breakdown and activate proteasomal function are poorly understood. Although PAN contains six identical ATPase subunits, we found that it exhibits three types of binding sites: 2 high affinity conformations (Kd=0.2uM), 2 with lower affinity (Kd=60uM), and 2 with conformations that fail to bind ATP. In fact, PAN never bound more than four of any type of nucleotide (ADP, ATP, or the nonhydroylyzable analog ATPγS), even at high concentrations. ATP binding to the high and lower affinity sites has distinct functional consequences on the proteasome. With two ATPγS molecules bound, PAN maximally stimulates opening of the gated channel for substrate entry into the 20S proteasome and has a high affinity for the 20S as well as for protein substrate. However, the binding of 4 ATPγS reduces PAN's ability to stimulate gate opening as well as its affinity for the 20S and substrates. These functional consequences of nucleotide binding are conserved in the mammalian 26S proteasome, since gate opening exhibited nearly identical multiphasic dependence on ATPγS concentration as found for the PAN‐20S complex. Because ATP binding drives the association of the C‐termini of the ATPase with the 20S and only two ATPase subunits bind ATP for maximal function it's likely that only two ATPases’ C‐termini dock into the 20S at any time and in a predictable pattern. This observation suggests how the hexameric ATPase ring associates with the heptameric 20S proteasome to regulate substrate degradation.

Assembly manual for the proteasome regulatory particle: the first draft

The proteasome is the most complex protease known, with a molecular mass of approx. 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the CP (core particle) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP-CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails, but are proximal to them. Some, but probably not all, of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.

Mechanisms of Force and Velocity Control in a Viral DNA Packaging Motor

dsDNA phages and viruses employ DNA packaging motors to translocate their genomes into small viral capsids against enormous internal pressures. Structural data, models, and sequence alignments have revealed the homology of critical putative functional domains of various nucleic acid translocases, including viral packaging motors, RNA helicases, and chromosome transporters. We used optical tweezers and mutational analysis to explore which functional domains of viral packaging motors govern their force generation and determine the velocities of packaging. A Q motif mutant of the phage λ DNA packaging motor, Y46F, was shown to have a decreased velocity (∼40% less than WT), increased slipping (∼10X WT), and steeper force-velocity dependence (∼6X WT), showing that the Q motif governs the force generation in translocation and DNA-motor interactions. In addition, we show that mutants with residue changes located in a previously undetermined domain of the motor, T194M and G212S, package dsDNA into viral capsids at ∼8X and ∼ 3X slower velocities than wild type (WT), respectively. Meanwhile a T194M pseudo-revertant (T194V) showed a near restoration of the WT velocity. The single molecule measurements of motor mutant translocation dynamics, genetic screening experiments, and structural modeling of ring ATPase dsDNA translocases suggest the location of a “velocity controller” domain within the phage λ packaging motor downstream the putative Walker B motif, which might be generalizable to other ring ATPase nucleic acid translocases. Importantly, this evidence may aid in explaining the different packaging rates of various dsDNA phages.

The crystal structure of apo -FtsH reveals domain movements necessary for substrate unfolding and translocation

The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo- and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.

Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions

Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C-termini a conserved hydrophobic-tyrosine-X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C-terminal HbYX motif and the 20S alpha-subunits and indicates that the intersubunit pocket in the 20S undergoes an induced-fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.

Receiver Domains Control the Active-State Stoichiometry of Aquifex aeolicus σ 54 Activator NtrC4, as Revealed by Electrospray Ionization Mass Spectrometry

A common challenge with studies of proteins in vitro is determining which constructs and conditions are most physiologically relevant. σ 54 activators are proteins that undergo regulated assembly to form an active ATPase ring that enables transcription by σ 54-polymerase. Previous studies of AAA + ATPase domains from σ 54 activators have shown that some are heptamers, while others are hexamers. Because active oligomers assemble from off-state dimers, it was thought that even-numbered oligomers should dominate, and that heptamer formation would occur when individual domains of the activators, rather than the intact proteins, were studied. Here we present results from electrospray ionization mass spectrometry experiments characterizing the assembly states of intact NtrC4 (a σ 54 activator from Aquifex aeolicus, an extreme thermophile), as well as its ATPase domain alone, and regulatory-ATPase and ATPase-DNA binding domain combinations. We show that the full-length and activated regulatory-ATPase proteins form hexamers, whereas the isolated ATPase domain, unactivated regulatory-ATPase, and ATPase-DNA binding domain form heptamers. Activation of the N-terminal regulatory domain is the key factor stabilizing the hexamer form of the ATPase, relative to the heptamer.

Insights into the molecular architecture of the 26S proteasome

Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the "base" part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the "wobbling" model that was previously proposed to explain the role of the regulatory complex in opening the gate in the alpha-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.

Hexameric assembly of the proteasomal ATPases is templated through their C termini

Substrates of the proteasome are recognized and unfolded by the regulatory particle (RP), then translocated into the core particle (CP) to be degraded1. A hetero-hexameric ATPase ring, containing subunits Rpt1-Rpt6, is situated within the base subassembly of the RP1. The ATPase ring sits atop the CP, with the Rpt C-termini inserted into pockets in the CP2–6. We have identified a novel function of the Rpt proteins in proteasome biogenesis through deleting the C-terminal residue from each Rpt. Our results indicate that assembly of the hexameric ATPase ring is templated on the CP. We have also identified an apparent intermediate in base assembly, BP1, which contains Rpn1, three Rpts, and Hsm3, a chaperone for base assembly. The Rpt proteins with the strongest assembly phenotypes, Rpt4 and Rpt6, were absent from BP1. We propose that Rpt4 and Rpt6 form a nucleating complex to initiate base assembly, and that this complex is subsequently joined by BP1 to complete the Rpt ring. Our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process.

Mechanism Of DNA Translocation By SpoIIIE/Ftsk

During cell division, chromosomal DNA is segregated both rapidly and specifically. In bacteria, chromosomal segregation is conducted by the DNA translocases FtsK (Escherichia coli) and SpoIIIE (Bacillus subtilis).These enzymes assemble at the division septum and translocate the chromosome (∼400 kb and ∼3 Mbp, respectively) unidirectionally by using the energy of ATP hydrolysis by means of their AAA+ motor domains. Despite functional and sequence similarities, SpoIIIE and FtsK were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here, we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to unveil the mechanism of DNA translocation by SpoIIIE, and to show that its wirestripping activity is largely responsible for compartment-specific gene expression during sporulation. We propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, and constitutes the first unified model for directional DNA transport by the SpoIIIE/FtsK/Tra family of AAA+ ring ATPases.

Intersubunit coordination in a homomeric ring ATPase.

Homomeric ring-ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the first direct observation of the inter-subunit coordination and the step size of such a ring-ATPase, the dsDNA packaging motor in the bacteriophage φ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 bp. Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated via a mechanism novel for ring-ATPases. In addition, a step size that is a non-integer number of base pairs demands new models for motor-DNA interactions.

Concealed enzyme coordination

Coordination between subunits is crucial for the proper functioning of multi-component molecular machines. A single-molecule study now allows glimpses into the mechanism used by subunits of one such machine. Homomeric ring ATPases are found in all forms of life and are involved in many processes such as chromosome segregation, protein unfolding and ATP synthesis. This remarkable functional diversity is generated by a single, highly conserved, structural core, responsible for the binding and hydrolysis of ATP. How these enzymes coordinate ATP hydrolysis and mechanistic function is largely unknown. One such ring ATPase from a bacteriophage, ϕ29, helps load the dsDNA genome into the viral shell. Moffitt et al. show that this five-membered motor packages the DNA in 10-base-pair bursts, each consisting of four individual 2.5-bp steps corresponding to the hydrolysis of a single ATP. Such a non-integral step size is unprecedented, and raises intriguing mechanistic questions about ATP hydrolysis within rings, and the interactions of the ring with DNA.

Structural basis of specific TraD–TraM recognition during F plasmid‐mediated bacterial conjugation

F plasmid-mediated bacterial conjugation requires interactions between a relaxosome component, TraM, and the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore. Here we present the crystal structure of the C-terminal tail of TraD bound to the TraM tetramerization domain, the first structural evidence of relaxosome-coupling protein interactions. The structure reveals the TraD C-terminal peptide bound to each of four symmetry-related grooves on the surface of the TraM tetramer. Extensive protein-protein interactions were observed between the two proteins. Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. Our results suggest that specific interactions between the C-terminal tail of TraD and the TraM tetramerization domain might lead to more generalized interactions that stabilize the relaxosome-coupling protein complex in preparation for conjugative DNA transfer.

Mechanism of Homotropic Control to Coordinate Hydrolysis in a Hexameric AAA+ Ring ATPase

AAA+ proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA+ characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA+ domain, PspF1–275, remodels the bacterial σ54–RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF1–275 indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent α-helical α/β subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA+ proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA+ ring. Homotropic coordination is functionally important to remodel the σ54 promoter. We propose a structural symmetry-based model for homotropic control in the AAA+ characteristic ring architecture.

Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to study the properties of DNA translocation by SpoIIIE in vitro and in vivo. These data allow us to propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.

Structural basis for the recognition between the regulatory particles Nas6 and Rpt3 of the yeast 26S proteasome

The 26S proteasome-dependent protein degradation is an evolutionarily conserved process. The mammalian oncoprotein gankyrin, which associates with S6 of the proteasome, facilitates the degradation of pRb, and thus possibly acts as a bridging factor between the proteasome and its substrates. However, the mechanism of the proteasome-dependent protein degradation in yeast is poorly understood. Here, we report the tertiary structure of the complex between Nas6 and a C-terminal domain of Rpt3, which are the yeast orthologues of gankyrin and S6, respectively. The concave region of Nas6 bound to the α-helical domain of Rpt3. The stable interaction between Nas6 and Rpt3 was mediated by intermolecular interactions composed of complementary charged patches. The recognition of Rpt3 by Nas6 in the crystal suggests that Nas6 is indeed a subunit of the 26S proteasome. These results provide a structural basis for the association between Nas6 and the heterohexameric ATPase ring of the proteasome through Rpt3.

The structural basis for regulated assembly and function of the transcriptional activator NtrC

In two-component signal transduction, an input triggers phosphorylation of receiver domains that regulate the status of output modules. One such module is the AAA+ ATPase domain in bacterial enhancer-binding proteins that remodel the sigma(54) form of RNA polymerase. We report X-ray solution scattering and electron microscopy structures of the activated, full-length nitrogen-regulatory protein C (NtrC) showing a novel mechanism for regulation of AAA+ ATPase assembly via the juxtaposition of the receiver domains and ATPase ring. Accompanying the hydrolysis cycle that is required for transcriptional activation, we observed major order-disorder changes in the GAFTGA loops involved in sigma(54) binding, as well as in the DNA-binding domains.