Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

A chemical genetics approach to examine the functions of AAA proteins.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

Structural snapshots of the cellular folded protein translocation machinery Bcs1.

Proteins destined to various intra- and extra-cellular locations must traverse membranes most frequently in an unfolded form. When the proteins being translocated need to remain in a folded state, specialized cellular transport machinery is used. One such machine is the membrane-bound AAA protein Bcs1 (Bcs1), which assists the iron-sulfur protein, an essential subunit of the respiratory Complex III, across the mitochondrial inner membrane. Recent structure determinations of mouse and yeast Bcs1 in three different nucleotide states reveal its homo-heptameric association and at least two dramatically different conformations. The apo and ADP-bound structures are similar, both containing a large substrate-binding cavity accessible to the mitochondrial matrix space, which contracts by concerted motion of the ATPase domains upon ATP binding, suggesting that bound substrate could then be pushed across the membrane. ATP hydrolysis drives substrate release and resets Bcs1 conformation back to the apo/ADP form. These structures shed new light on the mechanism of folded protein translocation across a membrane, provide better understanding on the assembly process of the respiratory Complex III, and correlate clinical presentations of disease-associated mutations with their locations in the 3D structure.

Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.

Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone.

The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.

Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes.

The Rea1 AAA+-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional ß-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.

Cryo-EM structure of the essential ribosome assembly AAA-ATPase Rix7.

Rix7 is an essential type II AAA-ATPase required for the formation of the large ribosomal subunit. Rix7 has been proposed to utilize the power of ATP hydrolysis to drive the removal of assembly factors from pre-60S particles, but the mechanism of release is unknown. Rix7's mammalian homolog, NVL2 has been linked to cancer and mental illness disorders, highlighting the need to understand the molecular mechanisms of this essential machine. Here we report the cryo-EM reconstruction of the tandem AAA domains of Rix7 which form an asymmetric stacked homohexameric ring. We trapped Rix7 with a polypeptide in the central channel, revealing Rix7's role as a molecular unfoldase. The structure establishes that type II AAA-ATPases lacking the aromatic-hydrophobic motif within the first AAA domain can engage a substrate throughout the entire central channel. The structure also reveals that Rix7 contains unique post-α7 insertions within both AAA domains important for Rix7 function.

X-ray structure of full-length human RuvB-Like 2 - mechanistic insights into coupling between ATP binding and mechanical action.

RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling.

Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13.

The maintenance of genome stability during mitosis is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex (APC/C, also known as the cyclosome)1,2. Unattached kinetochores control MCC assembly by catalysing a change in the topology of the ß-sheet of MAD2 (an MCC subunit), thereby generating the active closed MAD2 (C-MAD2) conformer3-5. Disassembly of free MCC, which is required for SAC inactivation and chromosome segregation, is an ATP-dependent process driven by the AAA+ ATPase TRIP13. In combination with p31comet, an SAC antagonist6, TRIP13 remodels C-MAD2 into inactive open MAD2 (O-MAD2)7-10. Here, we present a mechanism that explains how TRIP13-p31comet disassembles the MCC. Cryo-electron microscopy structures of the TRIP13-p31comet-C-MAD2-CDC20 complex reveal that p31comet recruits C-MAD2 to a defined site on the TRIP13 hexameric ring, positioning the N terminus of C-MAD2 (MAD2NT) to insert into the axial pore of TRIP13 and distorting the TRIP13 ring to initiate remodelling. Molecular modelling suggests that by gripping MAD2NT within its axial pore, TRIP13 couples sequential ATP-driven translocation of its hexameric ring along MAD2NT to push upwards on, and simultaneously rotate, the globular domains of the p31comet-C-MAD2 complex. This unwinds a region of the αA helix of C-MAD2 that is required to stabilize the C-MAD2 ß-sheet, thus destabilizing C-MAD2 in favour of O-MAD2 and dissociating MAD2 from p31comet. Our study provides insights into how specific substrates are recruited to AAA+ ATPases through adaptor proteins and suggests a model of how translocation through the axial pore of AAA+ ATPases is coupled to protein remodelling.