Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication.

ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1.

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1- DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

Cryo-EM structure of Vibrio cholerae aldehyde-alcohol dehydrogenase spirosomes.

Aldehyde-alcohol dehydrogenase (AdhE) is a metabolic enzyme and virulence factor in bacteria. E. coli AdhE (eAdhE) multimerizes into spirosomes that are essential for enzymatic activity. However, it is unknown whether AdhE structure is conserved in divergent bacteria. Here, we present the cryo-EM structure of AdhE (vAdhE) from Vibrio cholerae to 4.31 Å resolution. Overall, vAdhE spirosomes are similar to eAdhE with conserved subunit arrangement. However, divergences in key oligomerization residues cause vAdhE to form labile spirosomes with lower enzymatic activity. Mutating the vAdhE oligomerization interface to mimic eAdhE increases spirosome stability and enzymatic activity to levels comparable to eAdhE. These results support the generality of AdhE spirosome structures, and provide a structural basis to target vAdhE to attenuate bacterial virulence.

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.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human ß-cardiac myosin.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human ß-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human ß-cardiac myosin. We are using a recombinantly expressed human ß-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

The mechanism of dynein motility: insight from crystal structures of the motor domain.

Dynein is a large cytoskeletal motor protein that belongs to the AAA+ (ATPases associated with diverse cellular activities) superfamily. While dynein has had a rich history of cellular research, its molecular mechanism of motility remains poorly understood. Here we describe recent X-ray crystallographic studies that reveal the architecture of dynein's catalytic ring, mechanical linker element, and microtubule binding domain. This structural information has given rise to new hypotheses on how the dynein motor domain might change its conformation in order to produce motility along microtubules.

Crystal structure of the dynein motor domain.

Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein's large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ~300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an unusual asymmetric arrangement of ATPase domains in the ring-shaped motor domain, the manner in which the mechanical element interacts with the ATPase ring, and an unexpected interaction between two coiled coils that create a base for the microtubule binding domain. The arrangement of these elements provides clues as to how adenosine triphosphate-driven conformational changes might be transmitted across the motor domain.

Structure and functional role of dynein's microtubule-binding domain.

Dynein motors move various cargos along microtubules within the cytoplasm and power the beating of cilia and flagella. An unusual feature of dynein is that its microtubule-binding domain (MTBD) is separated from its ring-shaped AAA+ adenosine triphosphatase (ATPase) domain by a 15-nanometer coiled-coil stalk. We report the crystal structure of the mouse cytoplasmic dynein MTBD and a portion of the coiled coil, which supports a mechanism by which the ATPase domain and MTBD may communicate through a shift in the heptad registry of the coiled coil. Surprisingly, functional data suggest that the MTBD, and not the ATPase domain, is the main determinant of the direction of dynein motility.

Regulatory ATPase sites of cytoplasmic dynein affect processivity and force generation.

The heavy chain of cytoplasmic dynein contains four nucleotide-binding domains referred to as AAA1-AAA4, with the first domain (AAA1) being the main ATP hydrolytic site. Although previous studies have proposed regulatory roles for AAA3 and AAA4, the role of ATP hydrolysis at these sites remains elusive. Here, we have analyzed the single molecule motility properties of yeast cytoplasmic dynein mutants bearing mutations that prevent ATP hydrolysis at AAA3 or AAA4. Both mutants remain processive, but the AAA4 mutant exhibits a surprising increase in processivity due to its tighter affinity for microtubules. In addition to changes in motility characteristics, AAA3 and AAA4 mutants produce less maximal force than wild-type dynein. These results indicate that the nucleotide binding state at AAA3 and AAA4 can allosterically modulate microtubule binding affinity and affect dynein processivity and force production.