Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains

Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotide-regulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ～1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.     

Dynamics of alpha-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK(2). This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK(2)). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg(2+) and a MBP that is locked in a closed conformation induced a transition from open MalK(2) to semiopen MalK(2) without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK(2) therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK(2) opens, resetting the transporter in an inward-facing conformation.     

Anatomy of F1-ATPase powered rotation

F₁-ATPase, the catalytic complex of the ATP synthase, is a molecular motor that can consume ATP to drive rotation of the γ-subunit inside the ring of three αβ-subunit heterodimers in 120° power strokes. To elucidate the mechanism of ATPase-powered rotation, we determined the angular velocity as a function of rotational position from single-molecule data collected at 200,000 frames per second with unprecedented signal-to-noise. Power stroke rotation is more complex than previously understood. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur during the power stroke. The decreases in angular velocity that occurred with the lower-affinity substrate ITP, which could not be explained by an increase in substrate-binding dwells, provides direct evidence that rotation depends on substrate binding affinity. The presence of elevated ADP concentrations not only increased dwells at 35° from the catalytic dwell consistent with competitive product inhibition but also decreased the angular velocity from 85° to 120°, indicating that ADP can remain bound to the catalytic site where product release occurs for the duration of the power stroke. The angular velocity profile also supports a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F₁ structures 2HLD1 and 1H8E (Protein Data Bank).The purified F₁-ATPase is a molecular motor that can hydrolyze ATP to drive counterclockwise (CCW) rotation of the γ-subunit within the (αβ)₃-ring (Fig. 1A). In most living organisms, the F_o component of the F_oF₁ complex uses energy derived from a proton-motive force across a membrane to power F₁-dependent synthesis of ATP from ADP and P_i. Consumption of an ATP at each F₁ catalytic site, primarily composed of a β-subunit, correlates with a 120° rotational power stroke of the γ-subunit separated by a catalytic dwell with an 8-ms duration in Escherichia coli enzyme (1). A second “ATP-binding” dwell can occur after the γ-subunit has rotated ∼30° to 40° from the catalytic at limiting substrate concentrations (2, 3). Thus, three successive catalytic events that include power strokes and dwells are required to complete one revolution of the γ-subunit. Once bound to F₁, ATP is retained for 240° (3) such that the ADP and P_i generated are released two catalytic events later.Open in a separate windowFig. 1.Structural components of the F₁-ATPase molecular motor. (A) Top (from membrane) and side views of F₁ composed of the ring of α (orange) and β (purple) subunits surrounding the γ-subunit rotor (blue and green). (B) Open (β_E) and closed (β_D) conformations of the catalytic site composed of the catalytic domain (tan ribbon) and the lever domain (purple) relative to the γ-subunit coiled-coil (green) and foot (blue) domains. In the Gibbons et al. (32) F₁ structure (PDB ID code 1E79) used here, the γ-subunit foot domain is rotated 7° CCW from the structure determined by Menz et al. (4).The γ-subunit is composed of coiled-coil and globular “foot” domains where the former extends through the core of the (αβ)₃-ring (Fig. 1B). The β-subunits contain a catalytic domain and a C-terminal “lever” domain that is extended or open when the catalytic site is devoid of nucleotide and contracted (closed) when nucleotide is bound. In most F₁ crystal structures (4, 5), the coiled-coil faces the β-subunit with the open lever (β_E) whereas the foot domain extends over the lever domains of catalytic sites that usually contain bound ADP (β_D) and ATP (β_T). Although crystal structures provide excellent pictures of the subunit conformations at one rotational position, the rotational movement of the γ-subunit between these static structures and the mechanism in which ATP fuels this movement occurs remain major unresolved questions.Consensus is currently lacking regarding the relationship of nucleotide occupancy at the three catalytic sites to the catalytic dwell and ATP-binding dwell despite the intense scrutiny this question has received since Boyer and coworkers (6, 7) showed that F₁ operates via an alternating site mechanism. The catalytic dwell includes ATP hydrolysis and is believed to be terminated by the release of phosphate (3). Some single-molecule experiments support a mechanism whereby ATP binding and ADP release are concurrent during the ATP-binding dwell (3). As a result, only two catalytic sites are occupied the majority of the time such that three-site occupancy occurs transiently during the ATP-binding dwell. However, these results are inconsistent with the F₁ structure that contains transition state analogs and has three-site nucleotide occupancy (4). Nucleotide binding studies also strongly support a mechanism in which all three sites must be occupied (8, 9) and are consistent with other single-molecule studies that support alternative three-site mechanisms (10–12). At this time there is no consistent evidence that correlates any of the crystal structures to the prevailing rotational mechanism.The β-subunit lever domain is positioned to push against the γ-foot and the γ–coiled-coil as it opens and closes, respectively (Fig. 1B). The asymmetry of the γ-subunit at these interfaces resembles a camshaft that would be consistent with CCW directionality in response to lever movement. The energy for a 120° power stroke has been proposed to derive from the binding affinity of ATP that is used as ATP binding-induced closing of the β-lever (13) and is supported by experiments in which the lever was truncated (14).Based on single-molecule measurements, it was concluded that F₁ is nearly 100% efficient (15). A necessary outcome of this conclusion is that the 120° power strokes must occur at a constant angular velocity (13). Although a number of simulation studies have modeled rotation of the F₁-ATPase γ-subunit (16–19), only one of these (19) has provided a result showing that the angular velocity should vary during a power stroke. The claim of 100% efficiency (15) that serves as the energetic basis of this power stroke mechanism is unwarranted because the magnitude of the viscous flow coupling to the surface was unknown owing to technical limitations, and the authors erroneously used the value of ΔG° in lieu of ΔG in their calculation. The technical handicap was subsequently overcome by Junge and coworkers (20, 21), who relied on elastic probe curvature instead of rotation speed to calculate the average torque of the power stroke. Similar average torque values were subsequently obtained using rotation under limiting drag conditions, when the drag on the probe was measured directly (22).Based on 100% enzyme efficiency, it was difficult to explain how the energy from ATP binding was able to power 120° of rotation when the catalytic dwell interrupts rotation 80° after ATP binds. It was hypothesized that the remaining binding energy needed to power the final 40° of rotation until the next ATP binds is stored as elastic energy in the closed β-lever, which upon product release pushes on the γ-foot as it opens (13). However, to date, experimental evidence that tests these hypotheses is lacking owing to the inability to measure the rotary motion under conditions where the angular velocity is limited by the internal mechanism of the motor.Here we have resolved the angular velocity as a function of the rotational position using an assay that provides 10-μs time resolution. The results clearly show that the angular velocity is not constant during a power stroke, but undergoes a series of accelerations and decelerations as a function of rotational position. The slower angular velocity observed with the lower-affinity substrate ITP provides direct evidence that substrate binding affinity provides energy to power rotation. The correlation of the angular velocity profile of the final 85° of rotation presented here to the profile resulting from the simulations of Pu and Karplus (19) strongly supports a model in which ATP binding-dependent closure of the lever applies force to the γ-subunit. This also provides evidence that associates the F₁ structures of Kabaleeswaran et al. (23) and Menz et al. (4) with the protein conformations at 35° and 120° because they were used as the reference structures for the simulations of Pu and Karplus (19). The data also show that elevated ADP concentrations increase dwells at ∼35° and decrease the angular velocity between 85° and 120°. This indicates that ADP can remain bound subsequent to the ATP-binding dwell consistent with a three-site mechanism.     

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.     

Crystal structure of microsomal prostaglandin E2 synthase provides insight into diversity in the MAPEG superfamily

Prostaglandin E₂ (PGE₂) is a key mediator in inflammatory response. The main source of inducible PGE₂, microsomal PGE₂ synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design.     

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F₀F₁-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F₀F₁-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F₀F₁-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production.Mitochondria are involved in a wide range of cell functions, including fatty acid oxidation, calcium homeostasis, apoptosis, reactive oxygen species (ROS) signaling, and above all, production of ATP (1, 2). In neurons, these organelles are transported along neuronal processes to provide energy for areas of high energy demand, such as synapses (3). To support their functions, mitochondria exhibit a complex morphology consisting of separate and functionally distinct outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). The latter is structurally organized into two domains: an inner boundary membrane (IBM) and a cristae membrane (CM) (4). The current hypotheses imply that the morphology/topology of the IMM is tightly related to biochemical function, the energy state, and the pathophysiological state of mitochondria (5). Whereas the OMM contains porins [e.g., voltage-dependent anion channel (VDAC)], which mediate its permeability to molecules up to 10 kDa, the IMM topology is highly complex. It is comprised of different transport proteins, the ATP synthase (complex V), and complexes I, III, and IV of the electron transport chain, which are responsible for generating the proton motive force (pmf); pmf represents the driving force for not only ATP synthesis, but also other protein-mediated transport activities (for example, phosphate, pyruvate, and glutamate transport). Uncoupling protein 1 (UCP1; thermogenin), a member of the UCP subfamily, is known to dissipate the inner membrane proton gradient for heat production. One of the widely discussed functions for UCP4—another member of the same subfamily that is localized in neurons and neurosensory cells (6–9)—is the regulation of ROS by decreasing the pmf (10, 11). Although there is no unambiguous evidence revealing the exact UCP4 function, it was shown that UCP4 transports protons similar to UCP1 (12). It is, therefore, assumed that UCP4 and other UCPs possibly compete for protons with other proton-consuming proteins, including ATP synthase, but this phenomenon has not yet been studied in detail (13).Knowledge about exact protein localization at the mitochondrial inner membrane is of utmost importance for understanding the mechanisms behind the allocation of electrochemical potential to various demands, such as ATP production, thermogenesis, ROS regulation, etc. Because of resolution limitations, current data about IMM protein topography are scarce. By implementing immuno-EM, EM tomography, and live cell fluorescence microscopy, it was found that IBM and CM have different protein compositions (14–17). Few studies using superresolution microscopy have investigated nanoscale protein distribution, mainly focusing on respiratory chain proteins (18). In particular, there was strong evidence obtained in yeast, fibroblast-like COS cell line, and heart and liver mitochondria that ATP synthase and complexes I, III, and IV are mainly localized on the CM (14, 15, 19, 20). No data are available on the exact localization of UCPs along the IMM.In this study, we test the hypothesis that proton gradient-consuming proteins UCP4 and F₀F₁-ATP synthase are spatially separated within and/or between individual neuronal mitochondria. Therefore, we performed a two-color analysis of pairwise fluorescence-labeled mitochondrial proteins UCP4, VDAC, and F₀F₁-ATP synthase at 30 nm spatial resolution using superresolution imaging by direct stochastic optical reconstruction microscopy (dSTORM).     

Structural insight into signal conversion and inactivation by NTPDase2 in purinergic signaling

Cell surface-located nucleoside triphosphate diphosphohydrolases (NTPDase1, -2, -3, and -8) are oligomeric integral membrane proteins responsible for signal conversion and inactivation in extracellular nucleotide-mediated "purinergic" signaling. They catalyze the sequential hydrolysis of the signaling molecule ATP via ADP to AMP. Here we present the structure of the extracellular domain of Rattus norvegicus NTPDase2 in an active state at resolutions between 1.7 A and 2.1 A in four different forms: (i) apo form, (ii) ternary complex with the nonhydrolyzable ATP analog AMPPNP and cofactor Ca(2+), (iii) quaternary complex with Ca(2+) and bound products AMP and phosphate, and (iv) binary product complex with AMP only. Analysis of the ATP (analog) binding mode explains the importance of several residues for activity and allows suggestion of a catalytic mechanism. The carboxylate group of E165 serves as a catalytic base and activates a water molecule, which is well positioned for nucleophilic attack on the terminal phosphate. Based on analysis of the two product complex structures in which AMP adopts different conformations, a substrate binding mode for ADP hydrolysis is proposed. This allows for an understanding of how the same hydrolytic site can be engaged in ATP and ADP but not AMP hydrolysis.     

Direct observation of stepping rotation of V-ATPase reveals rigid component in coupling between Vo and V1 motors

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, V_o and V₁, and Enterococcus hirae V-ATPase (EhV_oV₁) actively transports Na⁺ in V_o (EhV_o) by using torque generated by ATP hydrolysis in V₁ (EhV₁). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV_oV₁ wild-type, aE634A, and BR350K mutants under various Na⁺ and ATP concentrations ([Na⁺] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na⁺] was low and [ATP] was high, under the condition that only Na⁺ binding to EhV_o is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhV_o rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na⁺], confirming that Na⁺ binding triggers the steps. When both [ATP] and [Na⁺] were low, under the condition that both Na⁺ and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhV_o and EhV₁, respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhV_o and EhV₁ do not share pausing positions, Na⁺ and ATP bindings occur at different angles, and the coupling between EhV_o and EhV₁ has a rigid component.

Rotary ATPases are ubiquitously expressed in living organisms and play important roles in biological energy conversions (1–6). These rotary ATPases are classified into F-, V-, and A-ATPases based on their amino acid sequences and physiological functions (6). Eukaryotic and bacterial F-ATPases (F_oF₁) and archaeal A-ATPases (A_oA₁) mainly function as ATP synthases driven by the electrochemical potential of ions across the cell membrane, although they can also act as active ion pumps driven by ATP hydrolysis depending on the cellular environment. In contrast, V-ATPases (V_oV₁) in eukaryotes primarily function as active ion pumps. V-ATPases are also found in bacteria, and some of them are termed V/A-ATPases based on their origin and physiological function in ATP synthesis (6–8).To date, numerous studies have been conducted to understand how the two motor proteins (i.e., F₁/A₁/V₁ and F_o/A_o/V_o) of the rotary ATPases couple their rotational motions and functions. Single-molecule studies using fluorescent probes (9–12), gold nanoparticle (AuNP) or nanorod probes (13–21), and Förster resonance energy transfer (16, 22, 23) have revealed the rotational dynamics of rotary ATPases for both ATP hydrolysis/synthesis directions. Furthermore, recent cryo–electron microscopic (cryo-EM) single-particle analyses have revealed entire architectures of the rotary ATPases with different structural states at atomic resolutions (24–35). In particular, several studies have demonstrated elastic coupling of F_oF₁ due to large deformations of the peripheral stalk connecting F_o and F₁ (25, 29, 35). However, few studies on other types of rotary ATPases with different functions and subunit compositions have been performed, and a comprehensive understanding of the energy transduction mechanism remains elusive.Enterococcus hirae V-ATPase (EhV_oV₁) works as an ATP-driven sodium ion (Na⁺) pump to maintain Na⁺ concentrations ([Na⁺]) inside the cell (Fig. 1A) (37–41). Note that we use the term V-ATPase or V_oV₁ because its physiological function is not ATP synthesis but active ion transport. EhV_oV₁ is a multisubunit complex composed of nine different subunits, namely, ac₁₀dE₂G₂ and A₃B₃DF complexes in EhV_o and EhV₁, respectively. In the EhV₁ A₃B₃DF complex, three pairs of the A and B subunits form a heterohexameric A₃B₃ stator ring, and the central rotor DF subcomplex is inserted into the A₃B₃ ring (Fig. 1B, Bottom) (42, 43). The EhV_o ac₁₀dE₂G₂ complex transports Na⁺ across the cell membrane. The membrane-embedded rotor ring is formed by a decamer of the tetrahelical transmembrane c subunit (c₁₀ ring; Fig. 1B, Top) connected with the central DF stalk via the d subunit (26, 44). The stator a subunit works as an ion channel, and two EG peripheral stalks interact with the a subunit and A₃B₃ ring to assure rotary coupling between EhV_o and EhV₁.Open in a separate windowFig. 1.(A) Overall architecture of EhV_oV₁. The dotted circular arcs represent the rotation direction driven by ATP hydrolysis. (B) (Top) Top view of the a subunit (cyan) and c₁₀ ring (brown) of EhV_o and (Bottom) A (yellow), B (orange), D (green), and F subunits (pink) of EhV₁. The black arrow in Top indicates the path of Na⁺ movement during ATP-driven rotation. The arcs in Bottom represent the catalytic AB pairs. (C) Side view of the a subunit viewed from the c subunit. This structure was constructed by the SWISS-MODEL server (36) using a structure of the a subunit of V-ATPase from T. thermophilus. The black arrows represent the path of Na⁺ movement during ATP-driven rotation. The mutated residue, aGlu634, is located on the surface of the entry half-channel of the a subunit as highlighted in red letters and a circle.In EhV₁, the ATP hydrolysis reaction is catalyzed at the interfaces of three A and B subunits. It drives a counterclockwise rotation of the DF rotor subunits as viewed from the EhV_o side (Fig. 1B, Bottom). Like other F₁/A₁/V₁ (11, 45), EhV₁ is a stepping motor that rotates 120° per one ATP hydrolysis (46). We previously revealed that the 120° step of isolated EhV₁ is further divided into 40 and 80° substeps by using high-speed and high-precision single-molecule imaging analysis with AuNP as a low-load probe (47). A main pause before the 40° substep involves ATP cleavage, phosphate release, and ATP binding events. The ATP binding triggers the 40° substep because the duration time is inversely proportional to [ATP]. The 80° substep is triggered by ADP release after a subpause with [ATP]-independent duration time. While the chemomechanical coupling scheme in EhV₁ has been revealed, because our previous single-molecule observation of EhV_oV₁ did not clearly resolve the pauses and steps (17), the elementary steps in the rotation of EhV_oV₁ have not been revealed.Although the mechanism of ion transport in F_o/A_o/V_o is not fully understood, the so-called “two-channel” model has been widely accepted (48–54). In this model, the a-subunit has two half-channels for ion entry/exit into/from the ion-binding sites of the rotor c ring. In the case of EhV_o, Na⁺ enters the half-channel from the cytoplasmic side and binds to the negatively charged Na⁺-binding sites of the c subunit (44, 55). Then, the charge-neutralized c subunit can move into the hydrophobic lipid membrane (53, 56). The rotational torque generated by ATP hydrolysis in EhV₁ is transmitted to EhV_o via the rotor d subunit, allowing the c₁₀ ring to rotate unidirectionally in the lipid membrane. Na⁺ translocated by a nearly single turn of the c₁₀ ring reaches another half-channel of the a subunit, which connects the Na⁺-binding site of the c subunit to the extracellular side. Then, Na⁺ is pumped out of the cell by a hydrated microenvironment (57) and/or electrostatic repulsion with the positively charged residues in the a subunit, aArg573 and aArg629, located at the interface between the two half-channels (Fig. 1C and SI Appendix, Fig. S1) (26). Because EhV_oV₁ has the c₁₀ ring, 10 Na⁺ are transported per single turn. Therefore, the step size of EhV_o is expected to be 36° (360°/10), similar to Escherichia coli and yeast F_oF₁, which also have c₁₀ rings (13, 14, 22, 35).The ion-to-ATP ratio is a central issue in the coupling mechanism of rotary ATPases. All known F₁/A₁/V₁ have three catalytic sites and threefold structural symmetry and hydrolyze or synthesize three ATP molecules per single turn. In contrast, the number of protomers forming the rotor c ring of F_o/A_o/V_o varies from 8 to 17 depending on the species, suggesting wide variations in the ion-to-ATP ratio of rotary ATPases (58, 59). In EhV_oV₁, because the rotor c ring of EhV_o has a 10-fold structural symmetry (Fig. 1B, Top), this enzyme has a structural symmetry mismatch and a noninteger ratio between transported Na⁺ and hydrolyzed ATP (10/3 = 3.3). If the rotational coupling between EhV_o and EhV₁ is elastic, as reported for E. coli and yeast F_oF₁, the symmetry mismatch is relieved by large deformations of the peripheral stalk and/or the central rotor (25, 29, 35, 60). On the other hand, if the coupling is rather rigid due to the multiple peripheral stalks of EhV_oV₁, the pausing positions of both EhV_o and EhV₁ would be observed independently in a single-molecule observation. To address this issue, it is required to directly visualize the rotational pauses and steps of EhV_oV₁ under conditions where the elementary steps of the rotation such as the bindings of Na⁺ and ATP to EhV_o and EhV₁, respectively, are both rate-limiting.Here we carried out high-speed and high-precision single-molecule imaging of the rotation of detergent-solubilized EhV_oV₁ by using 40-nm AuNP as a low-load probe. To resolve the rotational pauses and steps of EhV_o, a glutamate residue in the stator a subunit (aGlu634) was replaced with alanine. Since the mutated aGlu634 is located on the surface of the Na⁺ entry half-channel (Fig. 1C and SI Appendix, Fig. S2), Na⁺ binding to the c subunit in the EhV_oV₁(aE634A) mutant (hereinafter referred to as aE634A) is expected to become slower than in the wild type. The rotation rate of aE634A decreased about 10 times compared with that of the wild type, allowing us to clearly resolve the rotational pauses and steps in EhV_o. Under the condition that only Na⁺ binding is rate-limiting, aE634A showed 10 pausing positions per single turn and a step size of about 36°, consistent with 10 protomers in the c₁₀ ring of EhV_o. The duration time before the forward step was inversely proportional to [Na⁺], indicating that the dwell corresponds to the waiting time for Na⁺ binding. On the other hand, under the condition that both Na⁺ and ATP bindings are rate-limiting, 13 pausing positions per single turn were observed. Furthermore, backward steps smaller than 36° were occasionally observed only when ATP binding is also rate-limiting, indicating that EhV_oV₁ undergoes Brownian motion between adjacent pausing positions of EhV_o and EhV₁ when no torque is applied from EhV₁. Backward steps of 36° or larger than 36° were rarely observed, suggesting the suppression of reverse Na⁺ transport. Small backward steps were also frequently observed during three long ATP cleavage pauses of another mutant, EhV_oV₁(BR350K), in which ATP hydrolysis is rate-limiting for the rotation (47). From these results, we conclude that EhV_o and EhV₁ do not share their pausing positions, Na⁺ and ATP bindings occur at different angles, and their coupling has a rigid component.     

Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.     

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.Brown adipose tissue oxidizes fatty acids to produce heat for thermoregulation in the cold and is present in adult humans, where it holds promise in combating obesity (1, 2). Human brown fat depots correlate with leanness (3, 4) and, in mice, thermogenesis by brown fat has been shown to clear blood triglycerides and dispose of blood glucose, reducing metabolic disease (5). Thermogenesis by brown adipose tissue depends on uncoupling protein 1 (UCP1), a 33-kDa mitochondrial carrier protein that, when activated, transports protons across the mitochondrial inner membrane, decoupling electron transfer from ATP synthesis to dissipate the protonmotive force as heat. During the adrenergic stimulation of brown adipocytes (e.g., due to cold exposure), long chain fatty acids released from lipid droplets activate UCP1, overcoming the inhibition of the protein by cytosolic purine nucleotides, to induce thermogenesis (6). Therapeutically activating UCP1 directly, without the need for physiological stimuli, could provide treatments for metabolic disease (6, 7).The mechanism of proton conductance by UCP1 and the interplay that occurs between regulators is debated (refs. 8 and 9). Evidence largely from liposome studies has led to the proposals that fatty acid anions act as either cofactors, providing proton-binding sites in a transport channel within the protein (10), or transport substrates, which are exported by UCP1 and flip back across the inner membrane in a protonated state (11). Alternatively, patch-clamp studies with mitochondrial inner membranes suggest that either protonated or deprotonated fatty acid species can be transported by UCP1, which remain bound to the protein in the transport cycle to give a net proton transfer (9). During activation, fatty acids may compete with nucleotide binding directly, or indirectly through changes in the protein, as indicated by experiments with isolated mitochondria (12, 13). Claims of the involvement of other metabolites (e.g., ubiquinone-10 or 4-hydroxy-2-nonenal) in UCP1 activation have largely been refuted (14, 15).For many years, there has been a general consensus on the fundamental composition of UCP1. Following the observation that one nucleotide binds to two proteins (16), UCP1 was believed to be a dimer. This notion was supported by analytical ultracentrifugation (17), nucleotide binding (18–21), and protein cross-linking studies (18, 22), as well as many reports indicating that the related mitochondrial ADP/ATP carrier is dimeric too (see ref. 23 and references therein). Distinct from other carriers, however, UCP1 was found to function independently of cardiolipin. This mitochondrial lipid enhances the activity of many carriers (24) and can be observed bound to the ADP/ATP carrier in the available atomic structures (25–27). Measurements of ³¹P NMR have indicated that purified UCP1 preparations do not contain bound cardiolipin (28), in contrast to the three molecules reported to bind per ADP/ATP carrier (29), and reconstitution studies have indicated that the lipid is not required for UCP1 activity (e.g., refs. 30 and 31) but may alter the affinity of the protein for purine nucleotides (28).In recent years, several studies have demonstrated that the mitochondrial ADP/ATP carrier is monomeric, not dimeric (32–36), which raises questions on the oligomeric state of UCP1. Resolving the basic functional unit of UCP1 is paramount for understanding its mechanism. Here, we have developed novel methods to purify UCP1 from native sources, which have allowed us to reach new conclusions on the fundamental properties of the protein.     

Activation of pausing F1 motor by external force

A rotary motor F(1), a catalytic part of ATP synthase, makes a 120 degrees step rotation driven by hydrolysis of one ATP, which consists of 80 degrees and 40 degrees substeps initiated by ATP binding and probably by ADP and/or P(i) dissociation, respectively. During active rotations, F(1) spontaneously fails in ADP release and pauses after a 80 degrees substep, which is called the ADP-inhibited form. In the present work, we found that, when pushed >+40 degrees with magnetic tweezers, the pausing F(1) resumes its active rotation after releasing inhibitory ADP. The rate constant of the mechanical activation exponentially increased with the pushed angle, implying that F(1) weakens the affinity of its catalytic site for ADP as the angle goes forward. This finding explains not only its unidirectional nature of rotation, but also its physiological function in ATP synthesis; it would readily bind ADP from solution when rotated backward by an F(o) motor in the ATP synthase. Furthermore, the mechanical work for the forced rotation was efficiently converted into work for expelling ADP from the catalytic site, supporting the tight coupling between the rotation and catalytic event.     

DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.     

Structural study on the architecture of the bacterial ATP synthase Fo motor

We purified the F_o complex from the Ilyobacter tartaricus Na⁺-translocating F₁F_o-ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated F_o complex were present and in native stoichiometry (ab₂c₁₁). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c₁₁ rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c₁₁. A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the F_o stator, and a height profile reveals that it protrudes less from the membrane than c₁₁. The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c₁₁ helices. The location and distances of the stator helices impose spatial restrictions on the bacterial F_o complex.     

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation

F₁-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α₃β₃ ring. The crystal structures of F₁, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F₁ has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F₁, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F₁ from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F₁, the corresponding residues in the ADP-bound β (β_DP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F₁, α₃β₂β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F₁ showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F₁ paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that β_DP is the catalytically active form. The former point was again confirmed in experiments where F₁ rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F₁.     

The product of uncI gene in F1Fo-ATP synthase operon plays a chaperone-like role to assist c-ring assembly

Bacterial operons for F₁F_o-ATP synthase typically include an uncI gene that encodes a function-unknown small hydrophobic protein. When we expressed a hybrid F₁F_o (F₁ from thermophilic Bacillus PS3 and Na⁺-translocating F_o from Propionigenium modestum) in Escherchia coli cells, we found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI, c-subunits in F₁F_o existed as monomers but not as functional c₁₁-ring. When uncI was expressed from another plasmid at the same time, active F₁F_o with c₁₁-ring was produced. A plasmid containing only uncI and c-subunit gene produced c₁₁-ring, but a plasmid containing only c-subunit gene did not. Direct interaction of UncI protein with c-subunits was suggested from copurification of His-tagged UncI protein and c-subunits, both in the state of c₁₁-ring and c-monomers. Na⁺ induced dissociation of His-tagged UncI protein from c₁₁-ring but not from c-monomers. These results show that UncI is a chaperone-like protein that assists c₁₁-ring assembly from c-monomers in the membrane.     

Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.     

Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) P^G–O^3B bond cleavage; 3) four concomitant events: W1–PO₃⁻ formation, OH⁻ and proton cleavage, nucleophilic attack by the OH⁻ against P^G, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Actin, a major cytoskeletal protein, is an ATP-binding protein that exists in monomeric (G-actin) and filamentous (F-actin) forms. A large variety of cellular functions are driven by the cyclic processes of actin molecule assembly (polymerization) and disassembly (depolymerization). Actin ATP hydrolysis, which was originally discovered without knowing its biological significance (1), energetically drives the cyclic assembly–disassembly (2). Therefore, the elucidation of the mechanism of ATP hydrolysis is crucially important for our understanding of the cellular functions of actin.The actin molecule comprises two major domains, the outer domain (OD) and the inner domain (ID). An ATP molecule with a divalent cation binds in the cleft between the OD and ID (3). Upon incorporation into the actin filament, the actin molecule undergoes a conformational transition (4). In the monomeric G-form, the OD is twisted by about 20° relative to the ID, whereas in the filamentous F-form the two domains are almost flat.The entire cycle of actin assembly–disassembly proceeds in five sequential processes (below, each nucleotide tightly binds Mg²⁺, which is not explicitly indicated for simplicity. -G and -F indicate G-form and F-form actin, respectively):

• 1)ATP-G → ATP-F: The conformational transition, which is associated with the polymerization of the ATP-bound G-form actin.
• 2)ATP-F → ADP-Pi-F: The ATP hydrolysis reaction, triggered by process 1 with a rate constant of 0.3 s⁻¹ (5), which is considerably slower than other ATPases [e.g., myosin (20 to 200 s⁻¹) (6)]. The slow rate which allows further elongation, and the hydrolysis reaction occurs in the interior of the filament. The reaction is irreversible (7).
• 3)ADP-Pi-F → ADP-F: The Pi release, which occurs at an extremely slow rate with actin molecules in the interior of the filament [0.003 s⁻¹ (8) or 0.007 s⁻¹ (9)] but is much faster with molecules around barbed ends [>2 s⁻¹ (10) or 1.8 s⁻¹ (9)]. The Pi release is reversible. ADP-F has a modest affinity for Pi with a dissociation constant of 1 mM (9, 10), which is far below the cytosolic concentration, whereas ADP-G hardly binds Pi (∼60 mM) (10).
• 4)ADP-F → ADP-G: The depolymerization releases actin subunits at the ends of the actin filament.
• 5)ADP-G → ATP-G: The bound nucleotide exchanges from ADP for ATP, which occurs only in G-actin.

The cyclic reaction of barbed end assembly and the pointed end disassembly, occurring in actin alone at the critical concentration of monomers using ATP as an energy source, is called tread-milling (11). In the cytosol, where the actin concentration is far beyond the critical concentration for polymerization, cooperative behaviors of regulatory proteins accelerate the spontaneous tread-milling and/or facilitate filament turnover by promoting multiple reactions involving nucleation, capping, and severing, to drive fast cell migration (12, 13). Recent progress, particularly in kinetic analyses of single actin filaments, has revealed the contribution of the barbed end disassembly to monomer recycling (14, 15). Pi release dramatically alters the assembly properties of actin filaments in two ways: 1) by promoting spontaneous depolymerization and, more importantly, 2) by making the ADP-actin subunits more attractive to proteins that facilitate filament disassembly such as ADF/cofilin. Therefore, for comprehensively understanding of the actin cycle of assembly–disassembly, the mechanism and significance of the Pi release are of crucially important. This should be based upon our knowledge of the actin ATP hydrolysis process and the properties of ADP-Pi-F.Studies of the actin ATP hydrolysis mechanism have been hampered, mainly due to the lack of F-form actin structures at a sufficient resolution for identifying water molecules, which are the keys to the reaction. The crystal structure of the G-form actin is not suitable (3, 16), since the G-form actin is not ATP hydrolysis–competent (17). The cryogenic electron microscopy (cryo-EM) structures of the actin filament at over 3.1-Å resolutions (18–20) are also insufficient, because the details of the water molecules in the nucleoside binding cleft, particularly those surrounding the phosphate moiety, are missing. The actin filament has not been crystallized, because filaments with a fixed length have never been prepared. Previously, the ATP hydrolysis mechanism of F-form actin was studied by employing metadynamics simulations (21). However, the details of the reaction mechanism have remained obscure, since the simulations were based upon structural data that lacked water molecules (4).We now show that, in a 1:1 complex of the actin-binding protein fragmin domain-1 (F1) and actin (referred to as the F1A complex), the actin molecule is trapped in the F-form. This is the first monomeric actin structure in the F-form among the more than 260 actin structures deposited to date in the Protein Data Bank (PDB) (22). Fragmin is a member of the villin-gelsolin protein superfamily (23) and was first isolated from the slime mold Physarum polycephalum (24). Like gelsolin, fragmin severs F-actin and caps the barbed end in a Ca²⁺-dependent manner (24–26). Fragmin consists of three tandemly linked domains (F1, F2, F3) that are homologous to the N-terminal half of gelsolin (G1, G2, G3). G1 also forms a 1:1 complex with actin. However, in the G1–actin complex, the actin is in the G-form (27).The F1A complex provided 1.15-Å resolution structures of F-form actin binding AMPPNP, ADP-Pi, or ADP, with a magnesium ion at the catalytic site. Based on the high precision of the pre- and posthydrolysis structures, we performed quantum mechanics/molecular mechanics (QM/MM) calculations (28), which revealed the comprehensive reaction mechanism of the actin ATP hydrolysis. The results provide mechanistic answers to key questions regarding the cycle of actin state transitions: how the G-to-F conformational transition triggers ATP hydrolysis, why the ATP hydrolysis rate is much slower than those of other ATPases, why the hydrolysis is irreversible, why the Pi release is so slow, and why F-form actin is destabilized after the Pi-release.Our results demonstrate that, while some properties are quantitatively different, the overall catalytic strategy of actin ATP hydrolysis (up until the formation of ADP-Pi-F) is almost identical to those of P-loop type-motor proteins, such as F₁-ATPase (29), kinesin (30), and myosin (31–33), despite the substantial differences in the catalytic site structures. In contrast to the common ATP hydrolysis strategies, the processes after the hydrolysis are distinct in actin: The hydrolysis does not cause any overall conformational changes; therefore, the Pi is not promptly released and the ADP-Pi F-form actin structure is readily available. These observations have led us to propose hypotheses about how ATP hydrolysis and Pi release are related in general, and how Pi is released by actin in particular.     

Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin’s long-distance displacement or processivity by modulating the order–disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin’s ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/β-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Kinesin-1 is a motor protein that walks along microtubule (MT) filaments toward the plus-ends using energy acquired from the adenosine triphosphate (ATP) hydrolysis reaction while performing various cellular activities. For instance, it is responsible for the intracellular transport of vesicles, organelles, and signaling complexes (1–3). Neuronal kinesin KIF5A, for example (4, 5), is particularly important for retrograde axonal transport inside neurons. Several single-point mutations of the KIF5A kinesin are found to be extremely pathological, leading to a lethal neurodegenerative disease in humans, hereditary spastic paraplegia (HSP) disease (6, 7).Generally, kinesin motor proteins function in a homodimeric state. In earlier studies (8, 9), we have shown that all kinesins have some structurally important regions: motor domains which perform the catalytic conversion of ATP as well as binding to the MT, the coiled-coil stalk region that is essential for dimerization, and the neck linker region that connects the motor domain to the coiled-coil stalk region. It was shown that the energetic balance between kinesin binding to the MT and coiled-coil interactions in the dimerization interface is crucially important for the required order–disorder transition of the neck linker (8). This transition mediates the coordination between two motor domains of the kinesin dimer faithfully, which is required for a long-distance run on the MT. This process is referred to as processivity (10, 11). On the other hand, the rate of hydrolysis of ATP determines the gliding velocity of the kinesin on the MT. It is important to note here that the processivity, directionality of stepping, and gliding velocity are crucial for their function (7–12).Mutations related to HSP disease are genotypic. While most of these mutations are found to be in the motor domain of the kinesin, some are also found to be in the dimerization region. As shown in our earlier studies, which investigated the effect of HSP disease-related mutations on the dynamics of kinesin, these mutations are either at the MT-binding interface or at the dimerization region. Therefore, they destroy the energetic balance between the relative strength of these interactions, impairing the kinesin processivity that leads to HSP disease (8, 9). However, some other mutations in the motor domain are found not to affect the MT-binding strength but also lead to the disease. Experiments have shown that those mutations actually reduce the rate of ATP hydrolysis and thereby affect the gliding velocity of the kinesin (6, 7). It has been proposed that such kinesins, due to their sluggish movement, act as road blockers for other normally moving kinesis. In this article, we will concentrate on one of such mutations which is distal to the ATP hydrolysis reaction center.Like other motor proteins (13–25), the hydrolysis of the ATP molecule is an essential step in the mechanochemical cycle of kinesin. Here we focus on the mutation of the asparagine residue at a distal position with respect to the hydrolysis reaction center that is changed to a serine residue (Asn255Ser for PDB ID: 4HNA) (26). This mutation causes a reduction in the kinesin gliding velocity on the MT and in the ATPase rate compared to the wild-type one (8). The large distance (~11.5 Å) between the asparagine residue and the terminal γ-phosphorus atom of the ATP in the kinesin-1 structure suggests a long-distance or second sphere control of the ATP hydrolysis reaction (Fig. 1). Similar large distance effects have been previously observed. Biochemical studies have shown that the rate of ATP hydrolysis of kinesin-1 increases by ~33-fold upon binding to the MT (6). The MT-binding site and ATP hydrolysis reaction center are also far apart from each other (Fig. 1). Herein, using all-atom explicit solvent molecular dynamics (MD) simulations, hybrid quantum mechanics/molecular mechanics (QM/MM) methods, and an enhanced sampling approach for calculating free energies, we provide an explanation for the molecular origin of this second sphere control of the ATP hydrolysis reaction in kinesin-1, which is needed to understand its connection to HSP disease. In this connection, it is important to note that an earlier study (23) on this topic focusing on the ATP hydrolysis mechanism of kinesin did not consider the effect of MT binding and was limited to aqueous kinesin only. Inclusion of the MT-bound state is required to understand the complete process of ATP hydrolysis which is attempted in this study.Open in a separate windowFig. 1.ATP-bound kinesin-α/β-tubulin complex. It is composed of the kinesin motor domain region, α-tubulin, and β-tubulin. The tubulins are the units of the microtubule filaments. The γ-phosphate group of the ATP is surrounded by the Glu236, Arg203, Ser202, Thr92, Mg²⁺ ion, and six water molecules. The HSP disease-related residue, Asn255, is also shown, and the proposed second sphere interactions are highlighted.In this study, we will first present the three different reaction schemes for the ATP hydrolysis reaction in the wild-type kinesin bound to MT. We will then discuss our findings on the reaction mechanism and energetics for those three reaction schemes using hybrid QM/MM calculations and most importantly determine the pre-organization geometry for the reaction. Next, the effect of mutation and MT unbinding on the pre-organization geometry will be described using all-atom explicit solvent MD simulations and an enhanced sampling approach for calculating free energies. A multiple sequence alignment will also be presented to show how important are the residues/interactions of pre-organization geometry. Finally, we will discuss our results in the context of the HSP disease.     

The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating

The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a beta-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 A resolution, revealing a high-resolution image of its architecture formed by 19 beta-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The alpha-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.