Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Pro-Nifuroxazide Self-Assembly Leads to Triggerable Nanomedicine for Anti-cancer Therapy.

Transcription factor STAT3 has been shown to regulate genes that are involved in stem cell self-renewal and thus represents a novel therapeutic target of great biological significance. However, many small-molecule agents with potential effects through STAT3 modulation in cancer therapy lack aqueous solubility and high off-target toxicity, hence impeding efficient bioavailability and activity. This work, for the first time, reports a prodrug-based strategy for selective and safer delivery of STAT3 inhibitors designed toward metastatic and drug-resistant breast cancer. We have synthesized a novel lipase-labile SN-2 phospholipid prodrug from a clinically investigated STAT3 inhibitor, nifuroxazide (Pro-nifuroxazide), which can be regioselectively cleaved by the membrane-abundant enzymes in cancer cells. Pro-nifuroxazide self-assembled to sub 20 nm nanoparticles (NPs), and the cytotoxic ability was screened in ER(+)-MCF-7 and ER(-)-MD-MB231 cells at 48-72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide proliferation assay. Results indicated that Pro-nifuroxazide NPs are multifold more effective toward inhibiting cancer cells in a time-dependent manner compared to parent nifuroxazide. A remarkable improvement in the local concentration of drugs to as high as ∼240 fold when assembled into NPs is presumably the reason for this functional improvement. We also introduced molecular dynamics simulations to generate Pro-nifuroxazide nano-assembly, as a model assembly from triggerable anti-cancer drugs, to provide molecular insights correlating physicochemical and anti-cancer properties. In silico properties of Pro-nifuroxazide including size, chemistry of NPs and membrane interactions with individual molecules could be validated by in vitro functional activities in cells of breast cancer origin. The in vivo anti-cancer efficiencies of Pro-nifuroxazide NPs in nude mice xenografts with MCF-7 revealed remarkable growth inhibition of as high as 400%. Histopathological analysis corroborated these findings to show significantly high nuclear fragmentation and retracted cytoplasm. Immunostaining on tumor section demonstrated a significantly lower level of pSTAT-3 by Pro-nifuroxazide NP treatment, establishing the inhibition of STAT-3 phosphorylation. Our strategy for the first time proposes a translatable prodrug agent self-assembled into NPs and demonstrates remarkable enhancement in IC50, induced apoptosis, and reduced cancer cell population through STAT-3 inhibition via reduced phosphorylation.

Quenching protein dynamics interferes with HIV capsid maturation.

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA-SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA-SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix-coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

Detection of methylation on dsDNA using nanopores in a MoS2 membrane.

Methylation at the 5-carbon position of the cytosine nucleotide base in DNA has been shown to be a reliable diagnostic biomarker for carcinogenesis. Early detection of methylation and intervention could drastically increase the effectiveness of therapy and reduce the cancer mortality rate. Current methods for detecting methylation involve bisulfite genomic sequencing, which are cumbersome and demand a large sample size of bodily fluids to yield accurate results. Hence, more efficient and cost effective methods are desired. Based on our previous work, we present a novel nanopore-based assay using a nanopore in a MoS2 membrane, and the methyl-binding protein (MBP), MBD1x, to detect methylation on dsDNA. We show that the dsDNA translocation was effectively slowed down using an asymmetric concentration of buffer and explore the possibility of profiling the position of methylcytosines on the DNA strands as they translocate through the 2D membrane. Our findings advance us one step closer towards the possible use of nanopore sensing technology in medical applications such as cancer detection.

Skeletal Dysplasia Mutations Effect on Human Filamins' Structure and Mechanosensing.

Cells' ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered molecular dynamics simulations. The effects of skeletal dysplasia associated mutations of the structure and mechanosensing properties of Filamin were studied by combining various experimental and theoretical techniques. The results showed that Larsen syndrome associated mutations destabilize or even unfold domain 17. Interestingly, those Filamin functions that are mediated via domain 17 interactions with other proteins are not necessarily affected as strongly interacting peptide binding to mutated domain 17 induces at least partial domain folding. Mutation associated to Frontometaphyseal dysplasia, in turn, transforms 16-17 fragment from compact to an elongated form destroying the force-regulated domain pair.

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk.

The force-generating mechanism of dynein differs from the force-generating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-µs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.

Structural insights into the functional cycle of the ATPase module of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

Computational de novo design of antibodies binding to a peptide with high affinity.

Antibody drugs play a critical role in infectious diseases, cancer, autoimmune diseases, and inflammation. However, experimental methods for the generation of therapeutic antibodies such as using immunized mice or directed evolution remain time consuming and cannot target a specific antigen epitope. Here, we describe the application of a computational framework called OptMAVEn combined with molecular dynamics to de novo design antibodies. Our reference system is antibody 2D10, a single-chain antibody (scFv) that recognizes the dodecapeptide DVFYPYPYASGS, a peptide mimic of mannose-containing carbohydrates. Five de novo designed scFvs sharing less than 75% sequence similarity to all existing natural antibody sequences were generated using OptMAVEn and their binding to the dodecapeptide was experimentally characterized by biolayer interferometry and isothermal titration calorimetry. Among them, three scFvs show binding affinity to the dodecapeptide at the nM level. Critically, these de novo designed scFvs exhibit considerably diverse modeled binding modes with the dodecapeptide. The results demonstrate the potential of OptMAVEn for the de novo design of thermally and conformationally stable antibodies with high binding affinity to antigens and encourage the targeting of other antigen targets in the future. Biotechnol. Bioeng. 2017;114: 1331-1342. © 2017 Wiley Periodicals, Inc.

Detection and Mapping of DNA Methylation with 2D Material Nanopores.

DNA methylation is an epigenetic modification involving the addition of a methyl group to DNA, which is heavily involved in gene expression and regulation, thereby critical to the progression of diseases such as cancer. In this work we show that detection and localization of DNA methylation can be achieved with nanopore sensors made of two-dimensional (2D) materials such as graphene and molybdenum di-sulphide (MoS2). We label each DNA methylation site with a methyl-CpG binding domain protein (MBD1), and combine molecular dynamics simulations with electronic transport calculations to investigate the translocation of the methylated DNA-MBD1 complex through 2D material nanopores under external voltage biases. The passage of the MBD1-labeled methylation site through the pore is identified by dips in the current blockade induced by the DNA strand, as well as by peaks in the transverse electronic sheet current across the 2D layer. The position of the methylation sites can be clearly recognized by the relative positions of the dips in the recorded ionic current blockade with an estimated error ranging from 0% to 16%. Finally, we define the spatial resolution of the 2D material nanopore device as the minimal distance between two methylation sites identified within a single measurement, which is 15 base pairs by ionic current recognition, but as low as 10 base pairs by transverse electronic conductance detection, indicating better resolution with this latter technique. The present approach opens a new route for precise and efficient profiling of DNA methylation.

Chemomechanical Coupling in Hexameric Protein-Protein Interfaces Harnesses Energy within V-Type ATPases.

ATP synthase is the most prominent bioenergetic macromolecular motor in all life forms, utilizing the proton gradient across the cell membrane to fuel the synthesis of ATP. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, the precise molecular mechanism whereby vacuolar (V-type) ATP synthase fulfills its biological function remains largely fragmentary. Recently, crystallographers provided the first high-resolution view of ATP activity in Enterococcus hirae V1-ATPase. Employing a combination of transition-path sampling and high-performance free-energy methods, the sequence of conformational transitions involved in a functional cycle accompanying ATP hydrolysis has been investigated in unprecedented detail over an aggregate simulation time of 65 µs. Our simulated pathways reveal that the chemical energy produced by ATP hydrolysis is harnessed via the concerted motion of the protein-protein interfaces in the V1-ring, and is nearly entirely consumed in the rotation of the central stalk. Surprisingly, in an ATPase devoid of a central stalk, the interfaces of this ring are perfectly designed for inducing ATP hydrolysis. However, in a complete V1-ATPase, the mechanical property of the central stalk is a key determinant of the rate of ATP turnover. The simulations further unveil a sequence of events, whereby unbinding of the hydrolysis product (ADP + Pi) is followed by ATP uptake, which, in turn, leads to the torque generation step and rotation of the center stalk. Molecular trajectories also bring to light multiple intermediates, two of which have been isolated in independent crystallography experiments.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion.

The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane 'insertase' YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis.

Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter.

The widely conserved natural resistance-associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Mapping our cysteine accessibility scanning results onto this structure, we identified the metal-permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

Overall energy conversion efficiency of a photosynthetic vesicle.

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytb⁢c1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%-5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly.

UNLABELLED: Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection. IMPORTANCE: The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

Disulfide Bridges: Bringing Together Frustrated Structure in a Bioactive Peptide.

Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys(11)-Cys(23) and Cys(17)-Cys(29)) are anticorrelated within ∼1 µs of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. We propose that the native state contains structural elements that frustrate one another's folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys(4)-Cys(21) bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys(4)-Cys(21) bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys(4)-Cys(21) bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability.

Nascent peptide assists the ribosome in recognizing chemically distinct small molecules.

Regulation of gene expression in response to the changing environment is critical for cell survival. For instance, binding of macrolide antibiotics to the ribosome promotes translation arrest at the leader open reading frames ermCL and ermBL, which is necessary for inducing the antibiotic resistance genes ermC and ermB. Cladinose-containing macrolides such as erythromycin (ERY), but not ketolides such as telithromycin (TEL), arrest translation of ermCL, whereas either ERY or TEL stall ermBL translation. How the ribosome distinguishes between chemically similar small molecules is unknown. We show that single amino acid changes in the leader peptide switch the specificity of recognition of distinct molecules, triggering gene activation in response to ERY alone, to TEL alone or to both antibiotics or preventing stalling altogether. Thus, the ribosomal response to chemical signals can be modulated by minute changes in the nascent peptide, suggesting that protein sequences could have been optimized for rendering translation sensitive to environmental cues.

Trimodal Therapy: Combining Hyperthermia with Repurposed Bexarotene and Ultrasound for Treating Liver Cancer.

Repurposing of existing cancer drugs to overcome their physical limitations, such as insolubility, represents an attractive strategy to achieve enhanced therapeutic efficacy and broaden the range of clinical applications. Such an approach also promises to offer substantial cost savings in drug development efforts. Here we repurposed FDA-approved topical agent bexarotene (Targretin), currently in limited use for cutaneous manifestations of T-cell lymphomas, and re-engineer it for use in solid tumor applications by forming self-assembling nanobubbles. Physico-chemical characterization studies of the novel prodrug nanobubbles demonstrated their stability, enhanced target cell internalization capability, and highly controlled release profile in response to application of focused ultrasound energy. Using an in vitro model of hepatocellular carcinoma and an in vivo large animal model of liver ablation, we demonstrate the effectiveness of bexarotene prodrug nanobubbles when used in conjunction with catheter-based ultrasound, thereby highlighting the therapeutic promise of this trimodal approach.

Atomic Modeling of an Immature Retroviral Lattice Using Molecular Dynamics and Mutagenesis.

Defining the molecular interaction between Gag proteins in an assembled hexagonal lattice of immature retrovirus particles is crucial for elucidating the mechanisms of virus assembly and maturation. Recent advances in cryo-electron microscopy have yielded subnanometer structural information on the morphology of immature Gag lattices, making computational modeling and simulations feasible for investigating the Gag-Gag interactions at the atomic level. We have examined the structure of Rous sarcoma virus (RSV) using all-atom molecular dynamics simulations and in vitro assembly, to create the first all-atom model of an immature retroviral lattice. Microseconds-long replica exchange molecular dynamics simulation of the spacer peptide (SP)-nucleocapsid (NC) subdomains results in a six-helix bundle with amphipathic properties. The resulting model of the RSV Gag lattice shows features and dynamics of the capsid protein with implications for the maturation process, and confirms the stabilizing role of the upstream and downstream regions of Gag, namely p10 and SP-NC.

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.

The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

Mechanism of substrate translocation by a ring-shaped ATPase motor at millisecond resolution.

Ring-shaped, hexameric ATPase motors fulfill key functions in cellular processes, such as genome replication, transcription, or protein degradation, by translocating a long substrate through their central pore powered by ATP hydrolysis. Despite intense research efforts, the atomic-level mechanism transmitting chemical energy from hydrolysis into mechanical force that translocates the substrate is still unclear. Here we employ all-atom molecular dynamics simulations combined with advanced path sampling techniques and milestoning analysis to characterize how mRNA substrate is translocated by an exemplary homohexameric motor, the transcription termination factor Rho. We find that the release of hydrolysis product (ADP + Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition, the time scale seen also in experiment. The calculated free energy profiles and kinetics show that Rho unidirectionally translocates the single-stranded RNA substrate via a population shift of the conformational states of Rho; upon hydrolysis product release, the most favorable conformation shifts from the pretranslocation state to the post-translocation state. Via two previously unidentified intermediate states, the RNA chain is seen to be pulled by six K326 side chains, whose motions are induced by highly coordinated relative translation and rotation of Rho's six subunits. The present study not only reveals in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provides an effective approach to identify allosteric sites of multimeric enzymes in general.