Biology subjects

Hite, R. K.

Publications and source records attributed to Hite, R. K..

12 recordsLinked to original sources

bioRxiv2026

SLC33A1 exports oxidized glutathione to maintain endoplasmic reticulum redox homeostasis

The endoplasmic reticulum (ER) requires an oxidative environment to support the efficient maturation of secretory and membrane proteins. This is in part established by glutathione, a redox-active metabolite present in reduced (GSH) and oxidized (GSSG) forms. The ER maintains a higher GSSG:GSH ratio than the cytosol; however, the mechanisms controlling ER redox balance remain poorly understood. To address this, we developed a method for the rapid immunopurification of the ER, enabling comprehensive profiling of its proteome and metabolome. Combining this approach with CRISPR screening, we identified SLC33A1 as the major ER GSSG exporter in mammalian cells. Loss of SLC33A1 leads to GSSG accumulation in the ER and a liposome-based assay demonstrates that SLC33A1 directly transports GSSG. Cryo-EM structures and molecular dynamics simulations reveal how SLC33A1 binds GSSG and identify residues critical for its transport. Finally, an imbalance in GSSG:GSH ratio induces ER stress and dependency on the ER-associated degradation (ERAD) pathway, driven by a shift in protein disulfide isomerases (PDIs) toward their oxidized forms. Altogether, our work establishes SLC33A1-mediated GSSG export as a key mechanism for ER redox homeostasis and protein maturation.

cell biology↗

bioRxiv2026

Structural dynamics of adenine nucleotide potentiation of the human type 2 IP3 receptor

Inositol trisphosphate receptors (IP3R) are intracellular calcium (Ca2+) channels that mediate Ca2+ flux from the endoplasmic reticulum (ER) into the cytosol, playing a critical role in Ca2+ signaling. IP3R activity requires IP3 and Ca2+ and is potentiated by adenine nucleotides through a poorly understood mechanism. Here, we combined single-particle cryo-electron microscopy and all-atom molecular dynamics simulations to investigate the potentiation of IP3Rs by adenine nucleotides. Our structures reveal that ATP and cAMP bind to a conserved site in the juxtamembrane domain, which connects the cytoplasmic IP3- and Ca2+-binding sites with the transmembrane pore. Molecular dynamics simulations predict that the binding of adenine nucleotides rigidifies the juxtamembrane domain, primarily through the coordination of the adenine base. Consistent with the adenine base being critical for potentiation, mutations that disrupt the interactions with the adenine base perturb Ca2+ flux in cells. Taken together, our data suggest that adenine nucleotides potentiate IP3R channel activity by rigidifying the juxtamembrane domain to improve coupling between IP3 and Ca2+ binding and pore opening. Significance StatementInositol-1-4-5-trisphosphate receptors (IP3Rs) are the main intracellular calcium (Ca2+) release channels in non-excitable cells and contribute significantly to intracellular Ca2+ release in excitable cells. Regulation of IP3Rs by inositol-1-4-5-trisphosphate (IP3), adenine nucleotides, and Ca2+ is fundamental to both intracellular Ca2+ homeostasis and signaling. We show that adenine nucleotides modulate IP3R activity by tuning the dynamics of a mechanical fulcrum-like domain, the juxtamembrane domain (JD), which physically couples the IP3- and Ca2+-binding sites in the large regulatory cytoplasmic domain to the channel pore. Using a combination of structural, computational, and functional studies, we show that adenine nucleotides bind to and restrict the movement of the JD of the human type 2 IP3R (hIP3R2). We find that that the coordination of adenine nucleotides is primarily driven by hydrophobic interactions with the adenine moieties of the nucleotides and that these interactions are critical for normal hIP3R2 function. This work establishes the foundation for further research into the physiological role of adenine nucleotide modulation of IP3Rs.

biophysics↗

bioRxiv2025

Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P2

The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters, whose mutations are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here, we show that two related accessory {beta}-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, -4 and -5. Cryo-EM structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl- ion pathway. Unexpectedly, we find that PI(3,5)P2 stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PI(3,5)P2 collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.

cell biology↗

bioRxiv2024

Cryo-EM structures of G4-stalled CMG reveal inchworm mechanism of DNA translocation

DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we have characterized fork collisions with preformed G4s in the parental DNA using fully reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by blocking the CMG helicase. An ensemble of high-resolution cryo-EM structures of stalled yeast and human CMG complexes reveals that the G4 is fully folded and lodged inside the CMG central channel. The stalled CMG is conformationally constrained and arrests in the transition between translocation states. Unexpectedly, our analysis suggests that CMG employs an unprecedented inchworm mechanism to translocate on DNA. These findings illuminate the eukaryotic replication fork mechanism under both normal and perturbed conditions.

biochemistry↗

bioRxiv2023

Structural basis of lipid head group entry to the Kennedy pathway by FLVCR1

Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively1-6. Despite the importance of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here, we show that FLVCR1, whose mutation leads to the neurodegenerative syndrome PCARP7-9, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprised of aromatic and polar residues. Despite binding to a common site, the larger quaternary amine of choline interacts differently with FLVCR1 than does the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are critical for the transport of ethanolamine, while being dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLCVR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.

cell biology↗

bioRxiv2022

Structural titration reveals Ca2+-dependent conformational landscape of the IP3 receptor

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+-permeable cation channels whose biphasic dependence on cytoplasmic Ca2+ gives rise to cytosolic Ca2+ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP3R-mediated Ca2+ oscillations, the structural underpinnings of the biphasic Ca2+ dependence that underlies Ca2+ oscillations are incompletely understood. Here, we collected images of an IP3R with Ca2+ at concentrations spanning five orders of magnitude. Unbiased image analysis revealed that Ca2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for free energy, we demonstrate that Ca2+ binding at a high-affinity site allows IP3Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca2+, IP3Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca2+-dependence of IP3R channel activity.

biophysics↗

bioRxiv2022

Integrative genetic analysis identifies FLVCR1 as an essential component of choline transport in mammals

Genome-wide association studies (GWAS) of serum metabolites have the potential to uncover genes that influence human metabolism. Here, we combined an integrative genetic analysis associating serum metabolites to membrane transporters with a coessentiality map of metabolic genes. This analysis revealed a connection between feline leukemia virus subgroup C cellular receptor 1 (FLVCR1) - a plasma membrane protein - and phosphocholine, a downstream metabolite of choline metabolism. Loss of FLVCR1 in human cells and in mice strongly impairs choline metabolism due to a block in choline import. Consistently, CRISPR-based genetic screens identified several components of the membrane phospholipid machinery as synthetic lethal with FLVCR1 loss. Finally, cells lacking FLVCR1 exhibit mitochondrial defects and upregulate the integrated stress response (ISR) through heme regulated inhibitors kinase (HRI). Altogether, these findings identify FLVCR1 as a universal mediator of choline transport in mammals and provide a platform to discover substrates for unknown metabolite transporters.

cell biology↗

bioRxiv2022

Allosteric interactions prime androgen receptor dimerization and activation

The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

biochemistry↗

bioRxiv2022

Multistep loading of PCNA onto DNA by RFC

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3 end (3 ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATP{gamma}S and 3 ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

molecular biology↗

bioRxiv2021

Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175

Structures of the human lysosomal K+ channel TMEM175 in open and closed states revealed a novel architecture lacking the canonical K+ selectivity filter motif present in previously known K+ channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is capable of permeating K+ ions at the expected rate. Monovalent cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K+ over Na+, despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K+ channels.

biophysics↗

bioRxiv2021

Mechanisms of loading and release of the 9-1-1 checkpoint clamp

5 single-stranded/double-stranded DNA serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1s recruitment to 5 junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-RFC, in complex with 9-1-1 and a 5 junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1s recruitment to 5 junctions and reveal new principles of sliding clamp loading.

biophysics↗

bioRxiv2018

Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

neuroscience↗