Rahul Chadda

The insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog-specific functions despite high structural similarity. Each paralog contains six RNA-binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site-specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long-range arrangements of RNA-binding domains and linkers without altering the secondary structure, and with only modest effects on RNA-binding affinity. Critically, pSer-driven rearrangements occur both in the RNA-free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog-specific, phosphorylation-dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post-translational mechanism that tunes IGF2BP paralog dynamics across free and RNA-bound states to program target mRNA selection, processing, and translational fate.

Retroviral integration is mediated by viral integrase (IN), which synapses two viral long terminal repeat DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single-molecule Fӧrster resonance energy transfer, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous sarcoma virus intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature, functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation, and possibly a drug target for clinically relevant retroviruses.

The lysosome integrates anabolic signaling and nutrient sensing to regulate intracellular growth pathways. The leucine-rich repeat–containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signaling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show that LRRC8A affects leucine-stimulated mTOR; lysosome size; number; pH; expression of lysosomal proteins LAMP2, P62, and LC3B; and lysosomal function. Mutating an LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signaling and altered lysosomal morphology and pH observed in LRRC8A knockout cells. In vivo, LRRC8A-L706A;L707A knock-in mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance associated with reduced skeletal muscle PI3K-AKT-mTOR signaling, glucose uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8-mediated metabolic signaling function regulating lysosomal function, systemic glucose homeostasis, and insulin sensitivity.

Insulin secretion from pancreatic β cells is initiated by membrane potential depolarization, followed by activation of voltage-gated Ca ²⁺ channels to trigger Ca ²⁺ -mediated insulin vesicle fusion with the β cell plasma membrane. Here, we show that β cell swelling associated with glucose metabolism was sensed by LRRC8 channel complexes and contributed to insulin secretion. Hypertonic perfusate (360–380 mOsm) dose dependently impaired glucose-stimulated insulin secretion by counteracting β cell swelling. Hypotonic perfusate alone, independent of glucose stimulation or K _ATP channel closure, was sufficient to increase β cell intracellular Ca ²⁺ and trigger insulin secretion. Inhibition of sodium-potassium-chloride cotransporter-1 with bumetanide, which diminished the intracellular Cl ^– concentration in β cells and consequently reduced Cl ^– efflux via LRRC8 channel complexes, also significantly reduced hypotonic-stimulated insulin secretion. Finally, stimulation of insulin secretion by the glucokinase activator GKA50, which is known to induce β cell swelling, was entirely suppressed in β cell–targeted Lrrc8a KO islets. These data support a model wherein the LRRC8 channel complex senses β cell swelling triggered by glucose metabolism and regulates β cell insulin secretion through activation of LRRC8-mediated Cl ^– efflux.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido- L- phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. Highlights: RPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination. RPA is a ssDNA binding protein made of six domains situated across RPA70, RPA32 and RPA14 subunits. Four high affinity DNA binding domains engage the DNA. Site-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. Bulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro , Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo , Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl ^- /H ⁺ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect caused by their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium towards the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein’s biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties. IMPACT STATEMENT: Differences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

Measurements of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be described by the Poisson distribution. However, in reality, liposomes are heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the liposome occupancy distribution for membrane protein reconstitution while taking into account liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of liposomes with radius r = 200 nm, representing an idealization of vesicles extruded through 400 nm pores and compare it to the right-skewed distribution of 400 nm 2:1 POPE:POPG vesicles. As is the case for E. coli polar lipids, this synthetic composition yields a sub-population of small liposomes, ∼25 nm in radius with a long tail of larger vesicles. Previously published microscopy data of the co-localization of the CLC-ec1 Cl ^- /H ⁺ transporter with liposomes, and vesicle occupancy measurements using functional transport assays, shows agreement with the heterogeneous 2:1 POPE:POPG population. Next, distributions of 100 nm and 30 nm extruded 2:1 POPE:POPG liposomes are measured by cryo-electron microscopy, demonstrating that extrusion through smaller pores does not shift the peak, but reduces polydispersity arising from large liposomes. Single-molecule photobleaching analysis of CLC-ec1-Cy5 shows the 30 nm extruded population increases the ‘Poisson-dilution’ range, reducing the probability of vesicles with more than one protein at higher protein/lipid densities. These results demonstrate that the occupancy distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous populations with experimental knowledge of the liposome size distribution.