Biology subjects

Kitao, A.

Publications and source records attributed to Kitao, A..

7 recordsLinked to original sources

bioRxiv2026

Off-target-free chemogenetic platform that decodes physiological roles of target GPCRs

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A2A receptor (A2AR), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A2AR, we designed a prominent chemogenetic pair comprising a clinical A2AR antagonist and an engineered A2AR mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A2AR in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.

biochemistry↗

bioRxiv2026

A single conserved residue in FlhA couples export gate activation to substrate specificity switching

FlhA is a core component of the bacterial flagellar type III secretion system that functions as both an export engine and a switch regulator of substrate specificity. How these two processes are mechanistically coupled remains unclear. Here we identify a single conserved residue in FlhA, Arg-391, as a key molecular node that links export gate activation to substrate specificity switching. The R391A substitution blocks the transition from hook-type to filament-type secretion, resulting in polyhooks lacking filaments. An intermolecular salt-bridge formation of Arg-391 with Glu-351 of an adjacent FlhA subunit stabilizes the filament-type FlhA ring. Suppressor mutations in FlhA and the ruler protein FliK restore this interaction, indicating that FliK promotes the intermolecular salt-bridge formation during export switching. Moreover, Arg-391 is required for efficient ATPase-independent activation of the export gate. Together, these findings reveal an electrostatic mechanism by which FlhA coordinates export gate activation and substrate specificity switching during flagellar assembly.

microbiology↗

bioRxiv2026

Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice

Understanding brain function requires tools that allow precise manipulation of receptor signaling in defined cell types within intact neural circuits. Optogenetics and conventional chemogenetic approaches primarily enable cell-type-specific control of neuronal excitability using engineered receptors or ion channels. However, direct and reversible inhibition of endogenous neurotransmitter receptors in defined cell types has remained technically inaccessible. Here, we introduce native receptor-targeted chemogenetics (NARCH), a chemogenetic strategy that integrates structure-guided receptor engineering with allosteric ligand design to achieve reversible and temporally precise inhibition of endogenous receptor signaling with cell-type specificity in vivo. By applying NARCH to metabotropic glutamate receptor 1 (mGlu1), we demonstrate that mGlu1 signaling in cerebellar Purkinje cells is required for stabilization of motor learning across training sessions in freely moving mice. NARCH thus establishes a receptor-level chemogenetic framework for causal analysis of neural circuits and behavior.

neuroscience↗

bioRxiv2022

Conserved GYXLI motif of FlhA is involved in dynamic domain motions of FlhA required for flagellar protein export

Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side-chain interaction networks in FlhAC and promotes the cyclic open-close domain motions of FlhAC. The temperature- sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42{degrees}C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS. IMPORTANCEMany motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding-sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open- close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side-chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.

microbiology↗

bioRxiv2020

Regulation of caveolae through cholesterol-depletion dependent tubulation by PACSIN2/Syndapin II

The membrane shaping ability of PACSIN2 via its FCH-BAR (F-BAR) domain has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membrane contains abundant levels of cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. We show that the binding of PACSIN2 to the membrane could be negatively regulated by the amount of cholesterol in the membrane. We prepared a reconstituted membrane based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity to the F-BAR domain of PACSIN2 than the membrane without cholesterol, presumably due to a decrease in electrostatic charge density. Consistently, the acute depletion of cholesterol from the plasma membrane resulted in the appearance of PACSIN2-localized tubules with caveolin-1 at their tips, suggesting that the presence of cholesterol inhibited the prominent membrane tubulation by PACSIN2. The tubules induced by PACSIN2 were suggested to be an intermediate of caveolae endocytosis. Consistently, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the cells deficient in PACSIN2. These data suggested that PACSIN2 mediated the caveolae internalization dependently on the amount of cholesterol at the plasma membrane, providing a possible mechanism for the cholesterol-dependent regulation of caveolae.

cell biology↗

bioRxiv2019

High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration

In the chemotaxis of Escherichia coli, the cells behavioral switch involves binding of the phosphorylated form of the chemotaxis signaling protein CheY (CheYp) to the flagellar motor protein FliM, which induces the motor to rotate clockwise; otherwise, the motor rotates counterclockwise. To investigate high-pressure effects on CheYp-FliM binding at atomic resolution, we conduct molecular dynamics simulations of monomeric CheYp, the N-terminal fragment of the FliM (FliMN) that binds to CheYp, and the complex that forms between those proteins at pressures ranging from 0.1 to 100 MPa. The results show that the active form of monomeric CheYp is maintained even at 100 MPa but high pressure increases the water density in the first hydration shell and can cause conformational change of the C-terminal helix. The dissociation process of the complex is investigated by parallel cascade selection molecular dynamics (PaCS-MD), revealing that high pressure considerably induces water penetration into the complex interface. Pressure dependence of standard binding free energy calculated by the Markov state model indicates that the increase of pressure from 0.1 to 100 MPa weakens the binding by [~] 10 kcal/mol. Using high-pressure microscopy, we observed that high hydrostatic pressure reversibly fixes the motor rotation in the counter-clockwise orientation, which supports the notion that high pressure inhibits the binding of CheYp to FliM. We conclude that high pressure induces water penetration into the complex interface, which interferes with CheYp-FliM binding and prevents motor reversal.

biophysics↗

bioRxiv2018

An efficient timer and sizer of protein motions reveals the time scales of functional dynamics in the ribosome

The clock of life ticks as fast as how efficiently proteins could perform their functional dynamics. Protein complexes execute functions via several large-scale intrinsic motions across multiple conformational states, which occur at a timescale of nano-to milliseconds for well-folded proteins. Computationally expensive molecular dynamics (MD) simulation has been the only theoretical tool to time and size these motions, though barely to their slowest ends. Here, we convert a simple elastic network model (ENM), which takes a few seconds (ubiquitin) to hours (ribosome) for the analysis, into a molecular timer and sizer to gauge the slowest functional motions of proteins. Quasi-harmonic analysis, fluctuation-profile matching (FPM) and the Wiener-Khintchine theorem (WKT) are used to define the \"time-periods\", t, for anharmonic principal components (PCs) which are validated by NMR order parameters. The PCs with their respective \"time-periods\" are mapped to the eigenvalues ({lambda}ENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 86.9{lambda}ENM-1.9 and {sigma}2([A]2) = 46.1 {lambda}ENM-2.5 are established allowing the characterization of the time scales of Nuclear Magnetic Resonance (NMR)-solved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.\n\nAuthor SummaryThe time scale of biological processes is governed by protein functional dynamics that often corresponds to the largest conformational spread and the longest time scales among all possible motions. Current simulation methodologies cannot reach the slowest, often functional, motions especially for supramolecular machineries. Borrowing the spring-bead model used in polymer physics since 60s, the efficient elastic network model (ENM), introduced in 90s, captured all modes of protein motions but largely underestimated the time scales of slowest modes due to its harmonic approximation.\n\nHere we map water-damped modes sampled by MD simulations to corresponding ones in ENM and thereby establish 2 power laws that describe the authentic time scales and sizes for the slowest anharmonic modes as functions of ENM eigenvalues. With that, we adequately describe the sizes and time scales for three proteins, confirmed by NMR spectroscopy, and ribosome that contains ~0.2 million heavy atoms (~ 20 thousands coarse-grained nodes).

biophysics↗