Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F
            <sub>1</sub>
            –ATPase from bovine heart mitochondria

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107

The crystal structure has been determined of the F 1 -catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F 1 -ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the e-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the e-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α 3 β 3 -catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the e-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F 1 -ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated e-subunit unable to bind ATP, the enzyme remains inactive and the e-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the e-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α 3 β 3 -catalytic domain is likewise closely similar to those of active F 1 -ATPases. However, although the β E -catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Regulation of the thermoalkaliphilic F ₁ -ATPase from Caldalkalibacillus thermarum

Ferguson

Cook

Montgomery

et al. 2016

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107

“…Although conformational substeps have been observed structurally for superfamily 1 and 2 helicases and for motors such as the F1 ATPase, myosin, and kinesin (72)(73)(74)(75), similar insights have not been forthcoming for hexameric helicase/translocase systems. Functional substeps (as well as macrosteps) are frequently observed in single-molecule studies of motor proteins (76,77), but the architectural underpinnings of these events often remained unresolved.…”

Section: Structural Snapshots Reveal Translocation Substeps In a Hexamentioning

confidence: 99%

Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor

Thomsen

Lawson

Witkowsky

et al. 2016

Ring-shaped hexameric helicases and translocases support essential DNA-, RNA-, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the Escherichia coli Rho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering data show that Rho preferentially adopts an open-ring state in solution and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish structural snapshots of substepping events in a hexameric helicase/translocase.ATPase | helicase | motor protein | transcription | translocase H exameric helicases and translocases are motor proteins that play a central role in cellular transactions ranging from replication and repair to transcriptional regulation, chromosome packaging, and proteolytic homeostasis (1-4). Used to drive the processive and, at times, highly rapid movement of extended nucleic acid or protein chains through a central pore, ring-shaped motors face several challenges to their operation. One is that certain enzymes must transition through controlled ring opening and/or subunit assembly events to allow long, polymeric substrates that lack freely accessible ends to access interior motor elements (5-7). Another is that, once loaded, the molecular plasticity inherent to these assemblies must be harnessed to precisely coordinate ATP binding and hydrolysis between multiple subunits with the powering of substrate translocation, while at the same time alternating between tight and loose grips on the substrate to allow for processive movement. The substrate-dependent molecular rearrangements that underpin ring dynamics during these events remain poorly understood, not only for hexameric helicases and translocases, but for related ring-shaped switches as well.The Escherichia coli Rho transcription termination factor is a well-established model system for understanding hexameric translocase and helicase function (8, 9). During termination, Rho uses a cytosine-specific RNA-binding domain appended to the N terminus of a RecA-type ATPase fold (10, 11) to bind nascent RNA transcripts at cytosine-rich sequences [known as Rho utilization (rut) sites] (12, 13). Once loaded, Rho consumes ATP to translocate 5′→3′ toward a paused RNA polymerase, eventually promoting transcription bubble collapse and RNA release (14-19).Str...

“…These opening/closing and loosening/tightening motions in the αβ-dimer are closely related to chemical reactions, such as nucleotide binding and catalytic events (19,23,(45)(46)(47). For the systematic structural comparison, we selected five representative crystal structures: PDB ID codes 1BMF (4), 1H8E (48), 2JDI (38), 3OAA (41), and 4ASU (49). The 1BMF, 3OAA, and 2JDI structures have already been mentioned.…”

Section: Systematic Analysis Of Crystal Structures Of Atp Waiting Andmentioning

confidence: 99%

“…The 1BMF, 3OAA, and 2JDI structures have already been mentioned. The 1H8E and 4ASU structures are thought to be the catalytic intermediate states, particularly in the product release step (48,49). PCA identified two major axes, principal component 1 (PC1) and PC2, along which the structures can be best separated (Materials and Methods and Fig.…”

Section: Systematic Analysis Of Crystal Structures Of Atp Waiting Andmentioning

confidence: 99%

F ₁ -ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

Sugawa

Okazaki

Kobayashi

et al. 2016

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F 1 -ATPase (F 1 ) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α 3 β 3 , while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F 1 structure and the rotation angle of the motor. Remarkably, a structure of F 1 in an e-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F 1 -ATPase.