Γ-tubulin Ring Complex Research Articles

Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.

The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down γ -tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.

Versatile gamma-tubulin complexes contribute to the dynamic organization of MTOCs during Drosophila spermatogenesis

The initiation of microtubule formation is facilitated by γ-tubulin and γ-Tubulin Ring Complex (γ-TuRC) in various microtubule-organizing centers (MTOCs). While the heterogeneity of tissue-specific MTOCs and γ-TuRC in Drosophila testis has been described, their molecular composition and physiological significance are poorly understood. We investigated the testis-specific distribution and biochemical interaction of the canonical γ-TuRC proteins Grip163 and Grip84. We found that while Grip163 is present on the centrosome and basal body, Grip84 localizes to the centrosome and Golgi in spermatocytes and colocalizes with the testis-specific γ-Tubulin complexes (t-γ-TuC) at the basal body, apical nuclear tip, and near the elongated mitochondria after meiosis. We also showed the apical nuclear tip localization of some γ-TuRC interacting partners and proved their binding to t-γ-TuC proteins. These results highlight and prove the importance of the different γ-TuRCs in organizing the diverse MTOCs present during the extensive rearrangement of cell organelles during the spermatogenesis of Drosophila.

Partial closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation

Microtubule nucleation is templated by the γ-tubulin ring complex (γ-TuRC), but its structure deviates from the geometry of α-/β-tubulin in the microtubule, explaining the complex's poor nucleating activity. Several proteins may activate the γ-TuRC, but the mechanisms underlying activation are not known. Here, we determined the structure of the porcine γ-TuRC purified using CDK5RAP2's centrosomin motif 1 (CM1). We identified an unexpected conformation of the γ-TuRC bound to multiple protein modules containing MZT2, GCP2, and CDK5RAP2, resulting in a long-range constriction of the γ-tubulin ring that brings it in closer agreement with the 13-protofilament microtubule. Additional CDK5RAP2 promoted γ-TuRC decoration and stimulated the microtubule-nucleating activities of the porcine γ-TuRC and a reconstituted, CM1-free human complex in single-molecule assays. Our results provide a structural mechanism for the control of microtubule nucleation by CM1 proteins and identify conformational transitions in the γ-TuRC that prime it for microtubule nucleation.

CDK5RAP2 activates microtubule nucleator γTuRC by facilitating template formation and actin release

To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a template mechanism. However, its splayed structure does not match microtubule symmetry, leaving it unclear how γTuRC becomes an efficient nucleator. Here, we identify the mechanism of γTuRC activation by CDK5RAP2 centrosomin motif 1 (CM1). Using cryoelectron microscopy (cryo-EM), we find that activation involves binding of multiple CM1 dimers to five distinct sites around the outside of the γTuRC cone, which crucially depends on regulatory modules formed by MZT2 and the N-terminal extensions of GCP2 subunits. CM1 binding promotes lateral interactions between GCP subunits to facilitate microtubule-like conformations and release of luminal actin that is integral to non-activated γTuRC. We propose a model where generation of γTuRC with an expanded conformational range, rather than perfect symmetry, is sufficient to boost nucleation activity.

MZT2A serves as a prognostic biomarker and promotes the progression of kidney renal clear cell carcinoma

MZT2A is a novel core component in the γ-tubulin ring complex and aberrantly expressed in some types of tumors. However, MZT2A expression pattern across different cancers and its role in kidney renal clear cell carcinoma have not been sufficiently investigated. A thorough analysis of MZT2A expression landscape across 33 cancer types was conducted, utilizing 712 normal samples and 9807 tumor samples from TCGA (version 37.0), as well as 5112 normal samples from the GTEx databases. MZT2A's impact on KIRC cell viability and proliferation were evaluated through BrdU assays and monitored by cell imaging readers in MZT2A-expressing plasmid or siRNA-transfected cells. Additionally, the effects of MZT2A on cell apoptosis and cell cycle were detected using flow cytometry and Western blot. WGCNA analysis was employed to construct a co-expression gene network associated with MZT2A expression in KIRC, and Pearson correlation coefficient was utilized to examine the relationships between pairs of genes. MZT2A is overexpressed in 25 out of 33 types of cancer, including KIRC. In KIRC, high level of MZT2A was associated with higher clinical stage progression and poorer patients' survival. Downregulation of MZT2A decreased KIRC cell proliferation, retarded cell cycle and promoted apoptosis, while upregulation of MZT2A produced the opposite effects. WGCNA analysis and in vitro experiments revealed that MZT2A activated PI3K/AKT signaling pathway in KIRC. In all, MZT2A was overexpressed in most types of tumors. MZT2A served as an oncogene in KIRC and might be a potential target for guiding future treatments.

The structure of the γ-TuRC at the microtubule minus end - not just one solution.

In cells, microtubules (MTs) assemble from α/β-tubulin subunits at nucleation sites containing the γ-tubulin ring complex (γ-TuRC). Within the γ-TuRC, exposed γ-tubulin molecules act as templates for MT assembly by interacting with α/β-tubulin. The vertebrate γ-TuRC is scaffolded by γ-tubulin-interacting proteins GCP2-6 arranged in a specific order. Interestingly, the γ-tubulin molecules in the γ-TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ-TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ-TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α-tubulin mutant, resulted in a closed, cylindrical γ-TuRC showing canonical interactions between all γ-tubulin molecules and α/β-tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ-TuRC, with some γ-tubulin molecules failing to align with α/β-tubulin. This review discusses these outcomes, along with the broader implications.

Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules.

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the γ-tubulin ring complex (γ-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds γ-TuRC via γ-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from γ-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs' effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with γ-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215's C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in γ-TuRC templated nascent microtubules at cellular MTOCs.

γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology.

Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Structure of the native γ-tubulin ring complex capping spindle microtubules

Microtubule (MT) filaments, composed of α/β-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/β-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/β-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism. In this study, we determined cryo-electron microscopy structures of human γTuRC bound to a nascent microtubule. Structural changes of the complex into a closed conformation ensure that γTuRC templates the 13-protofilament microtubules that exist in human cells. Closure is mediated by a latch that interacts with incorporating tubulin, making it part of the closing mechanism. Further rearrangements involve all γTuRC subunits and the removal of the actin-containing luminal bridge. Our proposed mechanism of microtubule nucleation by human γTuRC relies on large-scale structural changes that are likely the target of regulation in cells.

SiRNA-Mediated BmAurora B Depletion Impedes the Formation of Holocentric Square Spindles in Silkworm Metaphase BmN4 Cells.

Silkworm ovary-derived BmN4 cells rely on chromatin-induced spindle assembly to form microtubule-based square mitotic spindles that ensure accurate segregation of holocentric chromosomes during cell division. The chromosome passenger protein Aurora B regulates chromosomal condensation and segregation, spindle assembly checkpoint activation, and cytokinesis; however, its role in holocentric organisms needs further clarification. This study examined the architecture and dynamics of spindle microtubules during prophase and metaphase in BmN4 cells and those with siRNA-mediated BmAurora B knockdown using immunofluorescence labeling. Anti-α-tubulin and anti-γ-tubulin antibodies revealed faint γ-tubulin signals colocalized with α-tubulin in early prophase during nuclear membrane rupture, which intensified as prophase progressed. At this stage, bright regions of α-tubulin around and on the nuclear membrane surrounding the chromatin suggested the start of microtubules assembling in the microtubule-organizing centers (MTOCs). In metaphase, fewer but larger γ-tubulin foci were detected on both sides of the chromosomes. This resulted in a distinctive multipolar square spindle with holocentric chromosomes aligned at the metaphase plate. siRNA-mediated BmAurora B knockdown significantly reduced the γ-tubulin foci during prophase, impacting microtubule nucleation and spindle structure in metaphase. Spatiotemporal BmAurora B expression analysis provided new insights into the regulation of this mitotic kinase in silkworm larval gonads during gametogenesis. Our results suggest that BmAurora B is crucial for the formation of multipolar square spindles in holocentric insects, possibly through the activation of γ-tubulin ring complexes in multiple centrosome-like MTOCs.

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis.

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.

The minus-end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules.

During mitosis, microtubules in the spindle turn over continuously. At spindle poles, where microtubule minus ends are concentrated, microtubule nucleation and depolymerization, the latter required for poleward microtubule flux, happen side by side. How these seemingly antagonistic processes of nucleation and depolymerization are coordinated is not understood. Here, we reconstitute this coordination in vitro combining different pole-localized activities. We find that the spindle pole-localized kinesin-13 KIF2A is a microtubule minus-end depolymerase, in contrast to its paralog MCAK. Due to its asymmetric activity, KIF2A still allows microtubule nucleation from the γ-tubulin ring complex (γTuRC), which serves as a protective cap shielding the minus end against KIF2A binding. Efficient γTuRC uncapping requires the combined action of KIF2A and a microtubule severing enzyme, leading to treadmilling of the uncapped microtubule driven by KIF2A. Together, these results provide insight into the molecular mechanisms by which a minimal protein module coordinates microtubule nucleation and depolymerization at spindle poles consistent with their role in poleward microtubule flux.

Autoinhibitory mechanism controls binding of centrosomin motif 1 to γ-tubulin ring complex.

The γ-tubulin ring complex (γTuRC) is the principal nucleator of cellular microtubules, and the microtubule-nucleating activity of the complex is stimulated by binding to the γTuRC-mediated nucleation activator (γTuNA) motif. The γTuNA is part of the centrosomin motif 1 (CM1), which is widely found in γTuRC stimulators, including CDK5RAP2. Here, we show that a conserved segment within CM1 binds to the γTuNA and blocks its association with γTuRCs; therefore, we refer to this segment as the γTuNA inhibitor (γTuNA-In). Mutational disruption of the interaction between the γTuNA and the γTuNA-In results in a loss of autoinhibition, which consequently augments microtubule nucleation on centrosomes and the Golgi complex, the two major microtubule-organizing centers. This also causes centrosome repositioning, leads to defects in Golgi assembly and organization, and affects cell polarization. Remarkably, phosphorylation of the γTuNA-In, probably by Nek2, counteracts the autoinhibition by disrupting the γTuNA‒γTuNA-In interaction. Together, our data reveal an on-site mechanism for controlling γTuNA function.