Janet G. Yang

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase–nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.

Dynamics of nucleosome remodelling by individual ACF complexes.

The ATP-dependent chromatin assembly and remodelling factor (ACF) functions to generate regularly spaced nucleosomes, which are required for heritable gene silencing. The mechanism by which ACF mobilizes nucleosomes remains poorly understood. Here we report a single-molecule FRET study that monitors the remodelling of individual nucleosomes by ACF in real time, revealing previously unknown remodelling intermediates and dynamics. In the presence of ACF and ATP, the nucleosomes exhibit gradual translocation along DNA interrupted by well-defined kinetic pauses that occurred after approximately seven or three to four base pairs of translocation. The binding of ACF, translocation of DNA and exiting of translocation pauses are all ATP-dependent, revealing three distinct functional roles of ATP during remodelling. At equilibrium, a continuously bound ACF complex can move the nucleosome back-and-forth many times before dissociation, indicating that ACF is a highly processive and bidirectional nucleosome translocase.

Structural Basis for Heavy Metal Detoxification by an Atm1-Type ABC Exporter

Although substantial progress has been achieved in the structural analysis of exporters from the superfamily of adenosine triphosphate (ATP)–binding cassette (ABC) transporters, much less is known about how they selectively recognize substrates and how substrate binding is coupled to ATP hydrolysis. We have addressed these questions through crystallographic analysis of the Atm1/ABCB7/HMT1/ABCB6 ortholog from Novosphingobium aromaticivorans DSM 12444, NaAtm1, at 2.4 angstrom resolution. Consistent with a physiological role in cellular detoxification processes, functional studies showed that glutathione derivatives can serve as substrates for NaAtm1 and that its overexpression in Escherichia coli confers protection against silver and mercury toxicity. The glutathione binding site highlights the articulated design of ABC exporters, with ligands and nucleotides spanning structurally conserved elements to create adaptable interfaces accommodating conformational rearrangements during the transport cycle. Structural and functional studies of ABC exporters provide insight into how glutathione derivatives are translocated. Crossing the Membrane Adenosine triphosphate (ATP)–binding cassette (ABC) transporters couple ATP hydrolysis to the translocation of a wide variety of substrates across cell membranes. Srinivasan et al. (p. 1137) describe the structure of a yeast mitochondrial transporter involved in Fe-S protein biogenesis. The structure reveals bound glutathione, which suggests that glutathione is part of the translocated substrate. J. Y. Lee et al. (p. 1133) describe the structure of a bacterial ABC transporter that confers protection against silver and mercury. This protein also binds glutathione derivatives. The structure provides insight into how ligand interactions are coupled to ATP hydrolysis.

The Rate of Bipolar Spindle Assembly Depends on the Microtubule-Gliding Velocity of the Mitotic Kinesin Eg5

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesins motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

Background: The MetNI transporter drives methionine import against its concentration gradient and regulates intracellular methionine levels. Results: Methionine is a noncompetitive inhibitor of MetNI ATPase activity, binding the transporter at two allosteric sites. Conclusion: MetNI regulates intracellular methionine concentrations via allosteric regulation. Significance: Regulation of methionine import at the protein level may minimize wasteful consumption of ATP when adequate intracellular supplies are available. The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.

The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex

Abstract Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nm. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ∼40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.

The Chromatin-Remodeling Complex ACF Functions as a Dimeric Motor to Space Nucleosomes

The chromatin structure at a given locus is a key determinant of its transcriptional state. Evenly spaced nucleosomes directly correlate with condensed chromatin structures and gene silencing. The ATP-dependent chromatin assembly factor (ACF) generates such structures in vitro and is required for transcriptional silencing in vivo. ACF generates and dynamically maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish such bidirectional movement is not known. Using FRET to follow disruption of histone-DNA interactions in real time we show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Employing Electron Paramagnetic Resonance (EPR) to resolve different populations of the nucleosome-ATPase complex, we find that the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Furthermore three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases bind facing each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome. Such a mechanism would allow rapid sampling of both sides of the nucleosome and allow bidirectional movement without dissociation. This novel dimeric motor mechanism differs from that of other dimeric motors such as kinesin and dimeric helicases that processively translocate in one direction and reflects the unique challenges faced by motors that move nucleosomes.