Dimerization of mammalian kinesin-3 motors results in superprocessive motion

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.Molecular motors of the kinesin and dynein superfamilies are responsible for a variety of microtubule-based intracellular functions such as vesicle transport, spindle assembly, and cytoskeletal organization. Several features of the kinesin and dynein mechanochemical cycles have been realized, yet much remains unknown (1, 2). For kinesin motors, much of the current knowledge is based on studies of kinesin-1, the founding member of the kinesin superfamily, where alternating catalysis by the two motor domains of the dimeric molecule results in unidirectional processive movement (the ability to take successive steps along the microtubule). Mechanochemical studies of other members of the kinesin superfamily have revealed interesting evolutionary adaptations to the kinesin motor domain that enable, for example, conversion of ATP hydrolysis into microtubule destabilizing activity (kinesin-8 and kinesin-13 families) (3, 4).The kinesin-3 family is one of the largest among the kinesin superfamily and consists of five subfamilies in mammals (KIF1, KIF13, KIF14, KIF16, and KIF28) (5). Kinesin-3 family members are characterized by high sequence conservation within their motor domains, a forkhead-associated domain, and in most cases a C-terminal lipid-binding domain such as a pleckstrin homology or Phox homology (PX) domain (5). The founding member of kinesin-3 family, CeUNC-104, was identified in Caenorhabditis elegans based on a mutation that severely affects the transport of synaptic vesicles to the axon terminal (6, 7). Since that time, mammalian kinesin-3 motors have been found to be associated with diverse cellular and physiological functions including vesicle transport (8–12), signaling (13, 14), mitosis (15–17), nuclear migration (18), viral trafficking (19, 20), and development (21). Defects in kinesin-3 transport have been implicated in a wide variety of neurodegenerative, developmental, and cancer diseases (22). However, a mechanistic understanding of this important class of cellular transporters is currently limited.To date, mechanistic studies have focused on truncated versions of mammalian KIF1A and its homolog CeUNC-104, and these studies have yielded contradictory results (23). For example, murine KIF1A has been proposed to function as a monomer that diffuses along the microtubule surface or as a processive dimer. Furthermore, dimerization has been proposed to occur before cargo binding or on the cargo surface (24–26). Thus, despite their widespread functions and clinical importance, the mechanisms of kinesin-3 motor regulation and motility remain largely enigmatic. Based on extensive characterization at the cellular and single-molecule levels of mammalian kinesin-3 motors from three subfamilies (KIF1, KIF13, and KIF16), we now provide a general model for kinesin-3 motor regulation and motility. We show that kinesin-3 motors are largely regulated by a monomer-to-dimer transition on the cargo surface that results in superprocessive motion for cargo transport.     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.There are more than 42 kinesin genes in the human genome, representing 14 distinct classes (1). All are members of the P-loop NTPase superfamily of nucleotide triphosphate hydrolases (2–4). Like other NTPases, kinesins share a conserved Walker motif nucleotide-binding fold (2, 4) that consists of a central twisted β-sheet and three nucleotide-binding loops, which are termed switch-1, switch-2, and the P-loop. Kinesins also share a common microtubule (MT) binding interface, which isomerizes between states that either bind MTs weakly or strongly, and a mechanical element, termed the neck linker (NL). The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5–7). NL isomerization (5, 8) is hypothesized to be the force-generating transition in kinesin motors (6, 7, 9–11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).Spectroscopic and structural studies have led to a model to explain how kinesins generate force (5–7, 9, 10, 12–15) (summarized in SI Appendix, Fig. S1), which proposes that the conformations of the nucleotide binding site, the MT-binding interface, and the NL are all determined by the state of the catalytic site. It predicts that when unbound to the MT, the motor contains ADP in its catalytic site and its NL is undocked. MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5, 7–10, 14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11, 14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as “open” and “closed,” and the NL alternates between docked and undocked (5, 6, 10, 13–15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11). However, the model fails to explain several features of kinesins. For example, it predicts that ATP does not bind to kinesin when the NL is docked. This prediction is inconsistent with studies of both Eg5 and kinesin-1, which suggest ATP binds more readily when the NL is docked (11, 16, 17). The model also predicts that the NL should be docked after ATP binding. However, electron paramagnetic resonance (EPR) probes attached to the NL show a significant population of both mobile and immobile NL states in the presence of both pre- and posthydrolytic ATP analogs (5). Furthermore, the model cannot explain the load dependence of stall, detachment, and back stepping, all of which require a branched pathway (11).To resolve these uncertainties, we have measured the kinetics of the structural changes that occur in switch-1 and the NL with nucleotide binding while the motor is bound to the MT in an experimental design that we refer to as “structural kinetics.” We carried out these experiments using an novel spectroscopic approach, termed transient time-resolved fluorescence resonance energy transfer, (TR)²FRET, that allows us to monitor the kinetics and thermodynamics of both the undocked/docked transition in the NL and the open/closed transition in switch-1 that accompany the process of nucleotide binding. These experiments explain differences in the enzymologies of kinesin-1 and Eg5 and suggest an interesting role for the L5 loop in controlling the timing of conformational changes in the Eg5 switch-1 and NL.     

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1–3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.Diffusion is a slow process that governs the overall speed of many biochemical and engineering processes. Diffusion is produced by random molecular motion (a “random walk”), and it leads to complete dispersion of particles but is inherently slow (1). Diffusion is, therefore, effective for small-length-scale applications but becomes impractical for applications requiring larger length scales. This is especially true when the sample contains dense architectures with small and tortuous pores that hinder molecular movement. Diffusion of molecules into and out of such a sample (e.g., fixed biological tissues) can take an impractically long time. For instance, it can take weeks for antibodies to diffuse a few millimeters into fixed tissues (2). The slow nature of diffusive transport has long limited the application of many existing and emerging techniques in biology and medicine to small or thin tissue samples (3–7).External forces can enhance transport of otherwise slowly diffusive molecules into and out of porous samples, but they have many limitations. For instance, hydrodynamic pressure can generate a convective flow across a porous sample (8), but the high pressure required to generate the flow can deform fragile samples such as soft tissues or polymeric materials (9). An electric field can drive electrophoresis of charged particles through a porous sample (10), but if the sample contains charged molecules, the electric field can also damage the sample. For this reason, electrophoresis may not be suitable for tissues or biomolecule–polymer hybrids containing charged endogenous biomolecules (11, 12). To avoid damaging samples, then, conventional chemical and biomedical methods for biological processing rely on the slow but safe diffusion method.However, with the development of in situ molecular interrogation methods (6, 13, 14) and tissue clearing techniques (2, 15–25) and an emphasis on studying organ-scale tissue as a whole, a pressing need has arisen for a means of expediting the transportation of various molecules into intact tissues. For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2, 15–25), but transporting these micelles into the intact tissue via diffusion can take weeks (2, 15). Although electrophoresis can speed up this process, as demonstrated in CLARITY, its application has been limited to low electric fields because using high fields can damage tissue structures (2). The problem is compounded by the fact that different regions of a tissue can have widely different electrical properties (26), leading to regions with concentrated electric fields. Electrophoresis, therefore, is ineffective for hastening transport of surfactant micelles into large, dense samples because only low electric fields can be used without risking damage to the sample.Faster transportation of molecular probes into intact tissue is also needed to reduce the time required to label large tissues. Diverse methods of tissue labeling are used in many areas of biological research and medical diagnosis for visualizing various biomolecules of interest. However, these techniques have been mostly confined to small samples owing to the difficulty of labeling and examining deep structures in large-scale intact tissues (6, 27–31). CLARITY and other emerging tissue-clearing techniques (2, 15–25) render intact tissues optically transparent and macromolecule-permeable, allowing examination deep inside a tissue with light microscopy, but staining such large samples remains challenging because diffusion of molecular probes is very slow; it can take several weeks to deliver molecular probes throughout a mouse-brain-sized tissue for staining (2). Therefore, it is imperative to develop a faster method for labeling thicker and denser tissues.Here we introduce a novel transport method termed stochastic electrotransport that rapidly and selectively disperses highly electromobile molecules in a porous sample without damaging the charged sample itself. We developed a computational model to theoretically demonstrate that a rotational electric field in a porous sample can enhance the apparent diffusivity of electromobile molecules with a quadratic dependence on their electromobilities. This electrophoretically driven diffusive transport selectively boosts the migration of freely moving molecules with high electromobility while suppressing the displacement of cross-linked endogenous biological molecules with low electromobility within the sample. We then developed an integrated platform to clear and stain intact tissues in record time using stochastic electrotransport. Our work demonstrates the potential of stochastic electrotransport to rapidly and nondestructively process large-scale intact biological systems with various biochemical techniques.     

Direct observation of electrogenic NH4+ transport in ammonium transport (Amt) proteins

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH₄⁺ scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH₄⁺/NH₃ transport is used instead in acid–base and pH homeostasis in kidney or NH₄⁺/NH₃ (and eventually CO₂) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.Ammonium transport (Amt) proteins are a class of trimeric, integral membrane proteins found throughout all domains of life. Despite moderate primary sequence homologies, distinct family members from bacteria, archaea, and eukarya (including humans) share conserved structural features and a high number of conserved amino acid residues that are considered functionally relevant (1–4). Although the involvement of all Amt proteins in transporting NH₄⁺/NH₃ across biological membranes is undisputed, their functional context is diverse. Prokaryotes and plants use Amt proteins to scavenge NH₄⁺/NH₃—a preferred nitrogen source for cell growth—from their environment, whereas mammals use Amt orthologs, the Rhesus proteins, for detoxification and ion homeostasis in erythrocytes and in the kidney and liver tissues (1, 5, 6).Three decades ago, Kleiner and coworkers suggested that Amt proteins are secondary active and electrogenic transporters for ammonium (7–9). Various groups have subsequently confirmed this finding by two-electrode voltage-clamp experiments with protein produced recombinantly from RNA injected into Xenopus laevis oocytes. Here, plant Amt and Rhesus proteins were the main object of study, but some mechanistic details remained unclear, in particular the distinction between electrogenic NH₄⁺ uniport (10–13), NH₃/H⁺ symport (11, 12), or electroneutral NH₄⁺/H⁺ antiport (14, 15). In contrast, bacterial Amt proteins were described as passive channels for the uncharged gas ammonia (NH₃) (16). The first crystal structure for an Amt family member, AmtB from Escherichia coli (17), was interpreted to support this hypothesis, and an ongoing controversy concerning the transported species has persisted in the field ever since. Several points have been raised to challenge the possibility of gas channeling, the most critical of which seems to be that at physiological pH the protonation equilibrium of NH₃—with a pK_a of 9.4—would be >99% on the side of charged NH₄⁺. This point implies that the import of neutral ammonia gas must be preceded by extracellular deprotonation and followed immediately by intracellular protonation. In summary, the import of NH₃ would thus result in a net NH₄⁺/H⁺ antiport. Such a mechanism would be electroneutral, but it would be secondary active in the presence of a proton motive force, resulting in a vectorial pumping of ammonium out of the cell—which is, of course, physiologically unreasonable. A second point is that biological membranes are themselves highly permeable for uncharged ammonia, with a permeability coefficient, P_d = 10⁻³ cm·s⁻¹, similar to that of water (18), such that a dedicated transport protein would hardly be required. Westerhoff and coworkers have argued that active Amt transport thus is imperative and that cells must be able to quickly block Amt transport upon intracellular accumulation of ammonium to avoid uncoupling of the proton gradient through back-diffusion of NH₃ (19). In prokaryotes and some plants, this blocking is the task of regulatory GlnK proteins belonging to the signal transducing P_II family that bind to corresponding ammonium transporters when their regulatory ligand 2-oxoglutarate, the primary metabolic acceptor for NH₄⁺ during nitrogen assimilation, is depleted (20).The high expectations to understand the mechanism of Amt transport from 3D structures have not been met to date. The available structures of E. coli AmtB (17, 21) and its complex with GlnK (22, 23) of A. fulgidus Amt-1 (24), Nitrosomonas europaea Rh50 (25, 26), and human RhCG (27) all show the same, inward-facing state of the protein. Such apparent structural rigidity would match the picture of a fast channel, whereas active transport is generally considered to involve conformational changes that expose a binding site for the cargo molecule(s) alternatingly to either side of the membrane (28). In addition, the difficulties to detect NH₄⁺/NH₃ and to assay Amt transport led to a lack of functional studies carried out in vitro on well-defined systems. An uptake assay with AmtB reconstituted in proteoliposomes was described to provide evidence for passive gas channeling (17), but the methodology was later contested (2). Assays based on the detection of radioactive methylammonium (MA) uptake were only carried out in whole cells of E. coli, and studies with voltage-clamp electrophysiology using Amt-1 reconstituted in planar lipid bilayers did not yield conclusive results (our work). A series of potentially important variants have been produced (29–39), but the lack of an adequate functional assay has precluded definite conclusions.The debate concerning the transport mechanism of Amt proteins has not been settled to date, necessitating a reliable functional in vitro assay. The finding that electrogenic transport was observed in X. laevis oocytes, but not in the far smaller membrane patch of a planar lipid bilayer setup, suggested that the transport rate of Amt proteins was possibly too low to lead to a detectable current response, unless a larger number of protein units were incorporated into the bilayer. We have therefore focused on a controlled method of in vitro electrophysiology that allows the simultaneous activation of >10⁸ protein units, the solid-supported membrane (SSM) electrophysiology (40). With this approach, pioneered by Fendler and coworkers, we were able to detect robust ion currents from isolated and reconstituted Amt proteins.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na⁺ symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na⁺-dependent. The transport activity of R262 mutants increased by 30–40% in the presence of a membrane potential, indicating substrate/Na⁺ cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1′ helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na⁺-coupled substrate symporters. We propose that a similar mechanism is operative in other Na⁺/H⁺-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.The carnitine/γ-butyrobetaine antiporter CaiT belongs to the betaine/carnitine/choline transporter family of secondary transporters that transfer substrates containing a quaternary ammonium group (1, 2) in and out of the cell. In Escherichia coli and other enterobacteria, such as Proteus mirabilis, carnitine is taken up by CaiT and converted to γ-butyrobetaine via the reaction intermediate crotonobetaine (3–5), which serves as an external electron acceptor under anaerobic growth conditions (4). Biochemical studies of E. coli CaiT (EcCaiT) have shown that it is a constitutively active, Na⁺/H⁺-independent antiporter (6).Crystal structures of CaiT from P. mirabilis (PmCaiT) and E. coli (EcCaiT) were recently determined with and without bound substrate (7, 8). These structures revealed a trimeric assembly of CaiT, as previously found (9). The protein was in an inward-facing conformation with two substrate molecules bound per EcCaiT monomer: one in the central transport site and another in an external binding site (7). Fluorescent binding assays with the protein reconstituted into liposomes indicated that substrate binding was cooperative. This suggested a regulatory role for the external binding site, which was proposed to increase substrate affinity and initiate substrate transport (7). Strikingly, the crystal structures revealed that CaiT adopts a fold similar to that of the LeuT-type transporters (7, 10, 11). This places it in the amino acid–polyamine-organocation (APC) superfamily (12), which shares a conserved architecture of two inverted repeats of five transmembrane (TM) helices each, implying common mechanistic principles. Among the APC transporters, the leucine transporter LeuT from the neurotransmitter/sodium symporter family (10), the betaine transporter BetP from the betaine/choline/carnitine transporter family (13), the benzyl-hydantoin transporter Mhp1 of the nucleobase/cation symport 1 family, and the Na⁺/galactose symporter vSGLT of the solute/sodium symporter family are substrate/sodium symporters (14, 15). Although the substrate to sodium stoichiometry varies for each Na⁺-dependent LeuT-type transporter, most of them possess a conserved sodium-binding site (Na2 site) at which the binding and dissociation of a sodium ion is proposed to facilitate structural changes that lead to substrate transport (16–18). Although an additional sodium-binding site (Na1 site) exists in transporters such as LeuT and BetP, the position or even the presence of this site is not strictly conserved among the Na⁺-dependent LeuT-type transporters (14, 15, 19–21).A small number of LeuT-type transporters, namely, AdiC (arginine/agmatine antiporter), ApcT (broad-specificity amino acid transporter), and CaiT, are Na⁺-independent (6, 22–25). The crystal structure of ApcT revealed that a lysine residue (K158) occupies a position equivalent to the Na2 site in LeuT. This lysine is proposed to undergo a protonation/deprotonation event that leads to conformational changes facilitating substrate transport (25). In CaiT, a methionine residue (M331) occupies a position equivalent to Na1 in LeuT, whereas a positively charged arginine residue (R262) occupies the Na2 site (7). Previously, we have shown that mutating M331 reduces transport activity but does not induce Na⁺ dependence in CaiT (7). Here we report that point mutations of R262 render CaiT inactive. Strikingly, the transport activity was partially restored by the addition of sodium, thus making these mutants Na⁺-dependent. Unlike wild-type, the transport activity of R262 mutants increased by 30–40% when a membrane potential was applied, suggesting that Na⁺ and substrate were cotransported. To find out whether and how the mutation affects the Na2 site, we determined the crystal structure of CaiT R262E. Although we did not find any major changes in the mutant protein, comparison with the substrate-bound EcCaiT wild-type protein revealed that the γ-butyrobetaine substrate adopts a different orientation at the central binding site, in which it directly interacts with the unwound part of TM1′. (To make the nomenclature consistent, we adopt the same helix numbering as in LeuT. Because CaiT has two extra helices at the N terminus in comparison with LeuT, TM3 in CaiT corresponds to TM1 in LeuT and is denoted as TM1′, and the remainder of the CaiT helices follow suit.) Because R262 is known to play a role in stabilizing the unwound part of TM1′ (7), we propose that R262 is crucial for substrate binding, similar to Na⁺ in the Na2 site of LeuT and BetP (17, 19). Indeed, our fluorescent binding assays using R262 mutants showed markedly decreased substrate affinity. Modeling CaiT in various conformations with BetP as a template revealed that R262 undergoes an oscillatory movement, contributing to different hydrogen bond networks in each conformation. We suggest that this movement of the positively charged R262 sidechain in CaiT mimics Na⁺ binding/unbinding in Na⁺-dependent LeuT-type transporters and plays a central role in the transport mechanism.     

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.The lactose permease of Escherichia coli (LacY) binds and catalyzes the coupled stoichiometric transport of d-galactose or β-d-galactopyranosides and H⁺ (galactoside/H⁺ symport) but does not interact with glucopyranosides. Biochemical studies (1–7) indicate that affinity and specificity are distinct properties determined by different interactions with LacY. Specificity is determined entirely by interactions with the galactopyranosyl ring, whereas affinity is better with α- than β-galactopyranosides (anomeric at C1) and can be increased dramatically by hydrophobic anomeric substituents with no effect on specificity.By using the free energy released from the energetically downhill movement of H⁺ in response to the electrochemical H⁺ gradient (∆µ̃_H+), LacY catalyzes uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H⁺ translocation is obligatory, in the absence of ∆µ̃_H+, LacY can also transduce the free energy released from the downhill transport of sugar to drive uphill H⁺ transport with the generation of ∆µ̃_H+, the polarity of which depends upon the direction of the sugar gradient (reviewed in refs. 8–10).Rates of equilibrium exchange and counterflow (exchange of one substrate molecule for another labeled molecule from the other side of the membrane) are unaffected by imposition of ∆µ̃_H+. Therefore, it is apparent that alternating accessibility of sugar- and H⁺-binding sites to either side of the membrane is the result of galactoside binding and dissociation and not ∆µ̃_H+ (reviewed in refs. 8–10). Moreover, downhill lactose/H⁺ symport from a high to a low lactose concentration in the absence of ∆µ̃_H+ exhibits a primary deuterium isotope effect that is not observed for ∆µ̃_H+-driven lactose/H⁺ symport, equilibrium exchange, or counterflow (11, 12). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (13, 14), whereas in the presence of ∆µ̃_H+, opening of a cavity on the other side of the membrane after dissociation of sugar and H⁺ is limiting (15). Based on these and other findings, a detailed mechanism for symport by LacY has been proposed (10).Initial X-ray structures of LacY without bound sugar exhibit two pseudosymmetrical bundles of mostly irregular transmembrane helices surrounding a large aqueous cavity in the middle of the molecule; these initial structures were open on the cytoplasmic side and sealed on the periplasmic side (an inward-open conformation) (16–19). However, our recent X-ray crystallography studies (20) of the conformationally trapped double-Trp mutant G46W/G262W cocrystallized with β-d-galactopyranosyl-1-thio-β-d-galactopyranoside (TDG) reveal an almost occluded conformation with a narrowly outward (periplasmic)-open conformation and a tightly sealed cytoplasmic side [Protein Data Bank (PDB) ID code 4OAA]. In addition, a molecule of TDG is bound in a central cavity. The evidence shows that specific galactoside binding is consistent with prior findings from mutagenesis (21–23) and uses induced fit to interact with the surrounding protein (20). The findings also provide a strong indication that the transport mechanism of LacY involves a substrate-bound, occluded, intermediate conformation.Lactose has only one galactopyranosyl ring. Similarly, one galactopyranosyl ring of TDG lies against Trp-151 (helix V), confirming hydrophobic stacking between the bottom of the galactopyranosyl ring and the aromatic indole ring as suggested (24, 25). Glu-269 (helix VIII) is the acceptor of hydrogen bonds from the C4-OH group of the galactopyranosyl ring (21, 26). The η1 NH₂ group of Arg-144 (helix V) donates a hydrogen bond to O5 in the ring and is within hydrogen-bond distance of the C6-OH, whereas the η2 NH₂ group of Arg-144 donates hydrogen bonds to the C2′-OH (the other galactopyranosyl ring) of TDG and to Glu-126 Oε2 (27–29). Glu-126 (helix IV) acts as hydrogen-bond acceptor from the C2′-OH of TDG (27–29). His-322 (helix X) acts as a hydrogen-bond donor/acceptor between the εNH of the imidazole ring and the C3-OH of TDG (29–33) and is stabilized by a hydrogen-bond donor/acceptor interaction with the δNH of the imidazole and the OH of Tyr-236 (29, 34, 35). Finally, Asn-272 (helix VIII) donates a hydrogen bond to the C4-OH of TDG (23). These interactions define the specificity of LacY (summarized in ref. 20).Cys-148 (helix V), well known with respect to substrate protection against alkylation (reviewed in ref. 22), is also close to bound TDG but not sufficiently close to interact directly. Similarly, replacement of Ala-122 (helix IV) with a bulky side chain or alkylation of A122C with bulky thiol reagents causes LacY to become specific for the monosaccharide galactose. Disaccharide binding and transport are blocked sterically (36). Although Ala-122 does not make direct contact with TDG, bulky substituents at position 122 would clearly impact disaccharide binding.p-Nitrophenyl-α-d-galactopyranoside (α-NPG) is a FRET acceptor from Trp-151 (37) and binds to LacY with ∼eight times higher affinity than the twofold symmetric TDG, ∼two orders of magnitude better than β-NPG, and ∼three orders of magnitude better than the physiological substrate lactose or the monosaccharide galactose (6, 7, 38, 39). As we show here, the side-chain interactions with the galactopyranosyl moiety of α-NPG that provide specificity are almost identical to those described for TDG. In contrast, the increased affinity of α-NPG versus TDG is probably attributable primarily to hydrophobic interactions between the nitrophenyl group of NPG and Phe-20, Met-23, and Phe-27 from helix I. In addition, the nitro group is in close contact with polar groups that can sustain polar interactions of the type that also pertain to the glucose moiety of lactose.     

Catalytically powered dynamic assembly of rod-shaped nanomotors and passive tracer particles

Nano- and microscale motors powered by catalytic reactions exhibit collective behavior such as swarming, predator–prey interactions, and chemotaxis that resemble those of biological microorganisms. A quantitative understanding of the catalytically generated forces between particles that lead to these behaviors has so far been lacking. Observations and numerical simulations of pairwise interactions between gold-platinum nanorods in hydrogen peroxide solutions show that attractive and repulsive interactions arise from the catalytically generated electric field. Electrokinetic effects drive the assembly of staggered doublets and triplets of nanorods that are moving in the same direction. None of these behaviors are observed with nanorods composed of a single metal. The motors also collect tracer microparticles at their head or tail, depending on the charge of the particles, actively assembling them into close-packed rafts and aggregates of rafts. These motor–tracer particle interactions can also be understood in terms of the catalytically generated electric field around the ends of the nanorod motors.The dynamic interactions between moving objects, in particular their response to external stimuli and their communication with each other, govern their collective behavior on many length scales. Schooling of fish and flocking of birds are good examples of emergent phenomena that are orchestrated by communication between individuals in a large group. In these systems, macroscale organization is typically driven by nearest neighbor interactions that follow simple rules. To reach the level of organization seen in such living assemblies, fast and precise (in terms of distances, angles, and velocities) communication and control are required from the members. It is now straightforward to create computational models from which such dynamic structures emerge, but artificial systems that mimic behaviors as complicated as fish schooling have very rarely been realized experimentally in macroscopic engineered systems (1). On the other hand, self-assembly at the nano- and molecular levels already demonstrates a certain level of complexity and has furthered our understanding of dynamic interactions at small scales (2, 3).There are already many examples of particle assembly driven by local forces or externally applied fields. Externally applied light, magnetic, electric, and acoustic fields can drive symmetric particles into ordered arrays (4–7). Colloidal Janus particles self-assemble into complex structures by various mechanisms (8–12). However, in these examples the particle aggregates hardly approach the complexity of assemblies of living organisms; the interactions are passive responses to local forces and external fields with very limited interparticle communication or active response to the behavior of nearest neighbors.Interactions between active particles, on the other hand, can more closely mimic those of living organisms (13–17). Powered particles generate signals, typically in the forms of chemical gradients, pressure, or electric potential, which can induce responses from nearby particles. When the particle density is high, collective behaviors can emerge. For example, rotating millimeter scale objects assemble into organized patterns (1, 18). Patterns also emerge in collections of dipolar disks that are mechanically propelled along their polar axis (19). Autonomously moving nano- and micromotors (20) exhibit rich collective behavior including swarming and schooling (21–27), predator–prey interactions (25), attraction and repulsion between rotors (28, 29), spatiotemporal oscillations (21, 25), and dynamic self-assembly (29, 30). Hydrophobicity and hydrodynamic interactions can also drive the assembly of nanomotors (31, 32). Although theoretical models and numerical simulations have furthered our understanding of these systems (33–37), there is still a lack of information on the pairwise interactions of particles that result in emergent behavior. Quantifying these interactions at the level of individual microparticles should lead to better understanding of active matter (whether it is composed of synthetic and biological micromotors) and may ultimately enable the prediction, design, and application of collective behavior.Here we report dynamic intermotor interactions and particle self-assembly in systems of self-electrophoretically driven platinum–gold nanorods. These catalytic nanomotors move autonomously at ∼20 μm/s when placed in 1–2 M H₂O₂ solution (38–40). In addition to their axial movement, which is well known from previous reports, we have observed that powered nanorods dynamically associate to form staggered doublets and triplets. When the nanomotors are mixed with charged tracer particles (the sizes of the motor and tracer particles are shown in Figs. S1–S4), they collect the passive particle “cargo” at the front or back end of the rods, depending on the charge on the passive particles, and drive their assembly into close-packed 2D rafts. None of these behaviors are observed with nanorods composed of a single metal. Analysis of tracking data and numerical simulations show that all of these behaviors originate from electrokinetic and electrostatic effects in systems of powered nanorods.     

Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter

Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H⁺-coupled polyspecific nutrient and drug exporter.Malaria caused by the protozoan parasite Plasmodium falciparum is one of the leading causes of mortality and morbidity in humans worldwide (1). Chloroquine (CQ) was initially a highly effective drug against this devastating disease (2). However, resistant strains of P. falciparum began to appear around 1950, and today practically all of the parasites are resistant to CQ (3–7). This has become a major threat to global public health. Extensive research identified a CQ transporter, P. falciparum CQ resistance transporter (PfCRT), which functions in resistant but not wild-type strains of the parasite (2, 8–14). The mutant transporter is expressed in the membranes of its digestive vacuoles (DV), excreting CQ from the vacuole and thus conferring resistance (15, 16). The decrease in intravesicular CQ concentration also promotes conversion of highly toxic hematin to hemozoin, generating resistance to other antimalarial drugs in addition to CQ (2, 9, 17–23). Therefore, it is important to clarify the transport mechanism of PfCRT to overcome drug resistance in malaria parasites (2, 9–11). However, the role of CQ-sensitive PfCRT transport under physiological conditions and how CQ-resistant PfCRT gains the ability to transport CQ remain unclear.Addressing the physiological relevance of PfCRT is a major issue in the area of infectious diseases. Attempts to obtain PfCRT-defective P. falciparum have been unsuccessful, suggesting that PfCRT is involved in DV transport processes that are essential for the parasites (2, 9). As CQ is a divalent amine that can freely penetrate through lipid membranes in its neutral form, but becomes impermeable upon protonation, we hypothesized that PfCRT recognizes amphipathic amines as transport substrates and acts as a polyspecific organic cation transporter. Similar to the vacuoles of yeasts and plants, the DV of the malaria parasite establishes a proton motive force or an electrochemical gradient of protons across the membrane as the sum of interior acidic pH gradient (ΔpH) and inside-positive membrane potentials (Δψ) by electrogenic proton pumps, vacuolar H⁺-ATPase, and vacuolar H⁺-pyrophosphatase to supply energy to secondary active transporters (23–27). Therefore, we also hypothesized that PfCRT may use the electrochemical gradient of protons as a driving force for transport.Recently, we have developed a transporter assay system that includes overexpression, purification, and reconstitution of eukaryotic transporters (28–30). The assay system enables us to study the mechanisms of action of transporters under defined ΔpH and Δψ. In the present study, we applied this technique to PfCRT to determine the transport properties of CQ-sensitive and CQ-resistant PfCRTs.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Regulation of OSR1 and the sodium,potassium, two chloride cotransporter by convergent signals

The Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and the STE20/SPS1-related proline-, alanine-rich kinase directly regulate the solute carrier 12 family of cation-chloride cotransporters and thereby modulate a range of processes including cell volume homeostasis, blood pressure, hearing, and kidney function. OSR1 and STE20/SPS1-related proline-, alanine-rich kinase are activated by with no lysine [K] protein kinases that phosphorylate the essential activation loop regulatory site on these kinases. We found that inhibition of phosphoinositide 3-kinase (PI3K) reduced OSR1 activation by osmotic stress. Inhibition of the PI3K target pathway, the mammalian target of rapamycin complex 2 (mTORC2), by depletion of Sin1, one of its components, decreased activation of OSR1 by sorbitol and reduced activity of the OSR1 substrate, the sodium, potassium, two chloride cotransporter, in HeLa cells. OSR1 activity was also reduced with a pharmacological inhibitor of mTOR. mTORC2 phosphorylated OSR1 on S339 in vitro, and mutation of this residue eliminated OSR1 phosphorylation by mTORC2. Thus, we identify a previously unrecognized connection of the PI3K pathway through mTORC2 to a Ste20 protein kinase and ion homeostasis.The protein kinases oxidative stress-responsive 1 (OSR1) and its homolog the STE20/SPS1-related proline-, alanine-rich kinase (SPAK or PASK) are the mammalian members of the germ-cell kinase VI subgroup of the large Ste20 branch of the mammalian kinome. OSR1 and SPAK directly regulate the solute carrier 12 family of cation-chloride cotransporters which modulate ion homeostasis throughout the body (1, 2). OSR1/SPAK kinase domains lie close to their N-termini and they contain two additional conserved regions named “PF1” and “PF2” [PASK and Fray (Drosophila homolog)] (3). PF1 is a C-terminal extension to the kinase domain and is required for enzyme activity (4). PF2 binds the consensus motif [(R/K)FX(V/I)] (5) in substrates including ion cotransporters and in regulators. OSR1 and SPAK are activated by with no lysine [K] (WNK) protein kinases, which phosphorylate the essential activation loop regulatory site as well as a second site in the PF1 region with an undefined function (6–9).The four WNK protein kinases are large enzymes notable for the alternative placement of the essential ATP-binding lysine residue in their catalytic domains, distinguishing them from other members of the protein kinase superfamily (10, 11). Initial attention was focused on these enzymes because certain mutations in two family members cause pseudohypoaldosteronism type II, a heritable form of hypertension (12). WNKs are activated by changes in tonicity. Cellular reconstitution studies and mouse genetics demonstrated the importance of WNK function in cell volume regulation and maintenance of blood pressure (13–19). Control of cation-chloride cotransporters through OSR1 and SPAK is among the best-documented actions of WNKs in diverse tissues (5, 20–22).WNKs also regulate serum- and glucocorticoid-inducible protein kinases (SGKs) through a noncatalytic mechanism leading to increased sodium influx through the epithelial sodium channel (ENaC) (23, 24). SGKs and the related Akt enzymes are activated by phosphorylation on multiple sites, most prominently a residue in the activation loop by the phosphoinositide-dependent protein kinase and on a second site in a C-terminal hydrophobic motif (25). The kinase that phosphorylates the hydrophobic motif site under many circumstances is the mammalian target of rapamycin complex 2 (mTORC2), which provides an additional phosphatidylinositol-3 kinase (PI3K)-dependent input to these kinases (26–33).In this study, we show that OSR1 is phosphorylated not only by WNKs but also on a C-terminal site, conserved in SPAK, by mTORC2. These studies reveal a link between WNK-OSR1/SPAK and the PI3K-mTORC2 cascade that suggests that OSR1 and SPAK integrate signals from osmosensing and survival pathways.     

Substrate-induced changes in the structural properties of LacY

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H⁺-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H⁺- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H⁺ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.The lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ (sugar/H⁺ symport) (3–6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H⁺ with the electrochemical H⁺ gradient (Δ$\tilde{\mu }$_H⁺; interior negative and/or alkaline). Conversely, because coupling between sugar and H⁺ is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H⁺ transport with the generation of Δ$\tilde{\mu }$_H+, the polarity of which depends upon the direction of the sugar concentration gradient (7–10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13–16). At the apex of the hydrophilic cavity, which is at the approximate middle of the molecule, the galactoside- and H⁺-binding sites are located. Side chains important for sugar recognition are located in both the N- and the C-terminal 6-helix bundles, whereas those involved in H⁺ binding are largely in the C-terminal 6-helix bundle. Most X-ray structures obtained thus far exhibit a tightly sealed periplasmic side with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm and an open cytoplasmic side (an inward-facing conformation). LacY is structurally highly dynamic, and binding of a galactoside closes the deep inward-facing cavity with opening of a complementary outward-facing cavity (reviewed in refs. 17, 18). Therefore, transport involves a large conformational change that allows alternating access of sugar- and H⁺-binding sites to either side of the cellular membrane, and a recent structure indicates that an occluded intermediate is involved (19). Although structural models of LacY provide insight into the conformational states involved in transport, a crystal structure represents a static structural snapshot, and therefore an understanding of how sugar binding triggers the cascade of events that results in dynamic alternating access remains unclear. Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20–25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26–35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic, and mechanical properties of the structural elements in a membrane protein in a physiologically relevant environment (20, 21).LacY binds galactopyranosides, and 4-nitrophenyl-α-d-galactopyranoside (αNPG) is among the lactose analogs with highest affinity (∼30 µM) (36). In the absence of substrate, LacY preferentially occupies an inward-facing open conformation, and substrate binding causes closing of the inward-facing cavity with opening of a reciprocal outward-facing cavity (reviewed in refs. 17, 18) with an occluded intermediate conformation (19). To understand the structural perturbations and properties associated with these conformations, we describe here the conformational, kinetic, energetic, and mechanical properties of LacY in the apo state and how these properties change upon substrate binding. SMFS and DFS are used to characterize the properties of individual structural segments of LacY and to describe how these regions change properties upon galactoside binding. To understand further how a single point mutation alters LacY, the conformationally restricted LacY mutant C154G (37), which crystallized originally (13), was also investigated. All measurements were conducted with wild-type (WT) or mutant C154G LacY embedded in a phospholipid membrane under physiological conditions. The findings quantify the structural properties of WT LacY, which change drastically upon sugar binding. In contrast, the structural properties of mutant C154G LacY remain largely unaffected by ligand binding.     

From the Cover: PNAS Plus: Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

Sec1/Munc18 (SM) family proteins are essential for every vesicle fusion pathway. The best-characterized SM protein is the synaptic factor Munc18-1, but it remains unclear whether its functions represent conserved mechanisms of SM proteins or specialized activities in neurotransmitter release. To address this question, we dissected Munc18c, a functionally distinct SM protein involved in nonsynaptic exocytic pathways. We discovered that Munc18c binds to the trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex and strongly accelerates the fusion rate. Further analysis suggests that Munc18c recognizes both vesicle-rooted SNARE and target membrane-associated SNAREs, and promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulation of fusion by Munc18c is specific to its cognate SNARE isoforms. Because Munc18-1 regulates fusion in a similar manner, we conclude that one conserved function of SM proteins is to bind their cognate trans-SNARE complexes and accelerate fusion kinetics. Munc18c also binds syntaxin-4 monomer but does not block target membrane-associated SNARE assembly, in agreement with our observation that six- to eightfold increases in Munc18c expression do not inhibit insulin-stimulated glucose uptake in adipocytes. Thus, the inhibitory “closed” syntaxin binding mode demonstrated for Munc18-1 is not conserved in Munc18c. Unexpectedly, we found that Munc18c recognizes the N-terminal region of the vesicle-rooted SNARE, whereas Munc18-1 requires the C-terminal sequences, suggesting that the architecture of the SNARE/SM complex likely differs across fusion pathways. Together, these comparative studies of two distinct SM proteins reveal conserved as well as divergent mechanisms of SM family proteins in intracellular vesicle fusion.The fusion of intracellular vesicles with target membranes requires two classes of conserved proteins: SNAREs and SM (Sec1/Munc18) proteins (1, 2). SNAREs are membrane-associated proteins that contain characteristic stretches of 60–70 amino acids known as core domains or SNARE motifs. Fusion is initiated when the core domains of the vesicle-rooted SNARE (v-SNARE) and the target membrane-associated SNAREs (t-SNAREs) zipper into a four-helix trans-SNARE complex between two apposed bilayers (2–5). N- to C-terminal zippering of the trans-SNARE complex brings the two membranes into close apposition to fuse (6–8).First isolated in genetic screens in yeast and nematodes (9, 10), SM proteins are hydrophilic factors of 60–70 kDa that regulate membrane fusion through binding to their cognate SNAREs (11–13). SM proteins exhibit a similar loss-of-function phenotype as that of SNAREs (i.e., abrogation of fusion) and are essential for every pathway of intracellular vesicle fusion (14–16). Mutations of SM proteins give rise to a number of human diseases, including epilepsy and inflammatory disorders, as well as arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome (17–21). Although the mechanism of SNAREs is well established, we are only beginning to understand how SM proteins regulate vesicle fusion.The best-characterized SM protein is the synaptic factor Munc18-1 (also known as nSec1 or STXBP1), which is required for the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane (1, 22). Synaptic neurotransmitter release serves as the nervous system’s major form of cell-to-cell communication and requires three SNARE proteins: syntaxin-1, SNAP-25, and VAMP2/synaptobrevin (3, 23, 24). Munc18-1 has been shown to play dual roles in synaptic vesicle fusion. First, Munc18-1 positively regulates the SNARE-dependent fusion reaction by interacting with the trans-SNARE complex and accelerating the fusion kinetics (12, 25–33). Second, Munc18-1 binds to syntaxin-1 monomer and locks the latter into a “closed” configuration that prevents SNARE complex formation (34–36). This closed syntaxin binding mode can promote syntaxin trafficking and guide the SNAREs down a specific assembly route with the assistance of Munc13 (27, 37–39). In view of the highly specialized nature of neurotransmitter release, however, it remains to be determined whether these functions constitute conserved mechanisms of the SM family proteins or represent specialized activities of Munc18-1 at the synapse. To address this question, it is imperative to dissect another member of the SM protein family and compare its functions with those of Munc18-1.In this study, we chose to characterize Munc18c (also known as Munc18-3), a ubiquitously expressed SM protein involved in nonsynaptic exocytic pathways (40, 41). Munc18c is not functionally interchangeable with the synaptic SM protein Munc18-1, indicating that they regulate distinct vesicle fusion pathways (16). Munc18c has been shown to regulate the exocytosis of the glucose transporter GLUT4 in body glucose homeostasis. Under basal conditions, GLUT4 is sequestered in intracellular vesicles in adipocytes and skeletal muscles. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, delivering GLUT4 to the cell surface to facilitate glucose uptake. GLUT4 vesicle fusion requires syntaxin-4 and SNAP-23 as the t-SNAREs, VAMP2 as the primary v-SNARE, and Munc18c as the cognate SM protein (40, 42). Mutations in Munc18c interfere with GLUT4 vesicle fusion and disrupt insulin-stimulated glucose transport into the cell (41, 43, 44). Importantly, Munc13 and synaptotagmins appear to be absent in adipocytes and are not known to be involved in the GLUT4 trafficking pathway (45, 46), highlighting major functional differences between GLUT4 exocytosis and synaptic release. In addition to GLUT4 exocytosis, Munc18c regulates a range of other exocytic pathways, including neutrophil secretion, amylase release, platelet exocytosis, and the sustained phase of insulin secretion (47–51).Although the physiological role of Munc18c in vesicle exocytosis is clear, its molecular mechanism remains to be established. Here, we sought to define the mechanisms underlying Munc18c function by reconstituting it into a defined fusion reaction containing GLUT4 exocytic SNAREs. We observed that Munc18c bound to the ternary trans-SNARE complex and strongly accelerated the fusion rate. Munc18c recognizes both the v- and t-SNAREs, and it potently promotes trans-SNARE zippering at the postdocking stage of the fusion reaction. The stimulatory activity of Munc18c was specific to the fusion reactions reconstituted with its cognate SNAREs. These data, in combination with previous findings of Munc18-1, suggest a conserved mechanism of SM proteins in intracellular vesicle fusion: to interact with their cognate trans-SNARE complex and accelerate the fusion kinetics. Like Munc18-1, Munc18c also binds to the syntaxin monomer. However, the binding of Munc18c to syntaxin did not block SNARE assembly or the fusion reaction, in agreement with our observation that six- to eightfold increases in Munc18c expression do not inhibit insulin-stimulated glucose uptake in adipocytes. These data indicate that Munc18c does not adopt the inhibitory closed syntaxin binding mode as shown for Munc18-1. Therefore, the closed syntaxin binding mode may not be a general feature of SM proteins. Unexpectedly, we found that the stimulation of fusion by Munc18c requires the N-terminal regions of the v-SNARE, whereas Munc18-1 recognizes the C-terminal motifs. These results suggest that although the conserved function of SM proteins involves binding to trans-SNAREs, the architecture of the SNARE/SM complexes likely differs across fusion pathways. Together, these findings establish the conserved as well as divergent functions of SM family proteins in intracellular vesicle fusion.     

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.Cell perception of extracellular stimuli is followed by a transient variation in cytosolic calcium concentration. Plants have evolved to produce the specific molecular machinery to interpret this primary information and to transmit this signal to the components that organize the cell response (1–4). The plant family of serine/threonine protein kinases PKS or CIPKs (hereinafter CIPKs) and their activators, the calcium-binding proteins SCaBPs or CBLs (hereinafter CBLs) (5, 6) function together in decoding calcium signals caused by different environmental stimuli. Available data suggest a mechanism in which calcium mediates the formation of stable CIPK–CBL complexes that regulate the phosphorylation state and activity of various ion transporters involved in the maintenance of cell ion homeostasis and abiotic stress responses in plants. Among them, the Arabidopsis thaliana CIPK24/SOS2-CBL4/SOS3 complex activates the Na⁺/H⁺ antiporter SOS1 to maintain intracellular levels of the toxic Na⁺ low under salt stress (7–9), the CIPK11–CBL2 pair regulates the plasma membrane H⁺-ATPase AHA2 to control the transmembrane pH gradient (10), the CIPK23–CBL1/9 (11, 12) regulates the activity of the K⁺ transporter AKT1 to increase the plant K⁺ uptake capability under limiting K⁺ supply conditions (12, 13), and CIPK23–CBL1 mediates nitrate sensing and uptake by phosphorylation of the nitrate transporter CHL1 (14). Together these findings show that understanding the molecular mechanisms underling CIPKs function provides opportunities to increase plant tolerance to abiotic stress and to improve plants for human benefit.CIPKs and CBLs contain discrete structural modules that are involved in the calcium-dependent regulation of the activity of the system and ensure the colocalization of the CIPK–CBL interacting pairs with their substrates at particular sites within the cell (15–17). CIPKs include an N-terminal kinase catalytic domain followed by a characteristic self-inhibitory motif known as FISL or NAF motif (hereinafter NAF, Pfam no. PF03822) (1, 6) and a protein phosphatase 2C binding domain designated as PPI (11, 18, 19). The NAF motif directly interacts with the catalytic domain and inhibits the kinase activity. The calcium-dependent interaction of CBLs with the NAF motif relieves the self-inhibition and activates the CIPKs (5, 6, 19, 20). The calcium binding to CBLs is mediated by four EF hand-like calcium binding motives. In addition, several CBLs are myristoylated and/or palmitoylated. These modifications are essential for recruiting their interacting CIPK partner to the plasma or vacuolar membrane (17, 21–23), and they may also be involved in the interaction of the CIPK–CBL complexes with their substrates (24). In addition, the phosphorylation of a conserved serine residue at the C terminus of CBLs by its interacting CIPK is required for activation of transporter substrates. It has been proposed that this process may stabilize the CIPK–CBL complex and trigger conformational changes to the binary complex that enhance its specificity toward target proteins (13, 25).Like many other kinases, CIPKs are also regulated by the phosphorylation of the activation loop by upstream kinases. This loop undergoes large conformational changes upon phosphorylation, allowing the entrance and the stabilization of substrates at the kinase active site (26). The activation loop of the CIPKs contains three conserved Tyr, Thr, or Ser residues. For some members of the family, the mutation of one of these residues to Asp mimics phosphorylation and produces the activation of the kinase, partly overcoming the effect of the self-inhibitory NAF motif. In fact, these phosphorylation-mimicking mutations and the deletion of the inhibitory domain produce a synergistic effect on the CIPK activity (6, 27–29). Transgenic plants expressing these CIPK24/SOS2 mutant proteins show improved salt tolerance (30).The kinase self-phosphorylation is another regulatory mechanism used by CIPKs. CIPK24/SOS2 is able to self-phosphorylate, and the autophosphorylation is important for its activity (31). Although the default state of CIPKs is inactive, some degree of autophosphorylation activity has been observed even for dephosphorylated and CBL-unbound CIPKs, which suggests that some CIPKs display basal activity (6). Indeed, it has been shown that the general regulatory factor 14-3-3 proteins (32) interact with CIPK24/SOS2 and repress its basal kinase activity when plants are grown in the absence of salt stress (33).The crystal structure of the binary complex of Ca²⁺-CBL4/SOS3 with the C-terminal regulatory moiety of CIPK24/SOS2 revealed the molecular mechanism underlying CBL-mediated activation of the CIPKs. The structure showed that the CIPK24/SOS2 self-inhibitory NAF motif is bound to CBL4/SOS3 and, consequently, it is not accessible to the kinase domain (19, 20). However, whether the CBL-unbound NAF blocks the active site or inhibits the enzyme by an allosteric mechanism is not known. To determine the molecular and structural basis for the CIPKs autoinhibition by the NAF and the activation by upstream kinases, we solved the structures of CIPK23 and CIPK24/SOS2. Our data show that inactivation of the kinases relies on the blockage of the active site by the NAF motif and the activation loop, which constitutes the basis for the conserved NAF-mediated self-inhibition of the CIPKs.