Biology subjects

Southworth, D. R.

Publications and source records attributed to Southworth, D. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Biased stochastic motor dynamics drive bidirectional, processive translocation by the AAA+ disaggregase Hsp104.

Protein aggregation disrupts proteostasis and drives neurodegeneration. Hsp104 is a hexameric, ring-shaped AAA+ ATPase that dissolves protein aggregates, yet how hexamers translocate and extract polypeptides trapped in mechanically resistant aggregates remains unclear. Using substrates that recapitulate the physical constraints of aggregates, we establish that Hsp104 is a processive, bidirectional translocase that can dynamically switch direction while threading a single polypeptide. On mechanically restrained substrates and prions, Hsp104 hexamers execute biased stochastic transitions among three conformational states at individual interprotomer interfaces: closed, extended, and a previously unobserved hyperextended form. These transitions follow kinetically favored paths rather than a rigid rotary sequence. The resulting biased stochastic stepping, enabled by the conformational plasticity of Hsp104 hexamers, underpins operational adaptability and redefines the functional logic of AAA+ motors.

biochemistry↗

bioRxiv2026

Phosphorylation of S396 and S400 Promotes Tau Self-Assembly and Favors the Chronic Traumatic Encephalopathy (CTE) Protofilament Fold

Tau hyperphosphorylation is linked to tauopathy aggregates, but the effects of individual sites on tau assembly remain unclear. Here, we used protein semisynthesis to generate defined Tau(297-407) proteoforms containing specific combinations of phosphorylation within the PHF1 epitope. Spontaneous aggregation revealed that phosphorylation of S396, and S400 to a lesser extent, promoted nucleation, while phosphorylation of T403 and S404 suppressed assembly. A similar reactivity trend of pS396 > pS400 > WT > pT403 > pS404 was observed for seeded assembly, and all proteoforms, even anti-aggregation ones, were incorporated. The fibrils have similar thermodynamic stability, suggesting that phosphorylation selectively tunes reactivity and not thermodynamics. Cryo-EM revealed that pS400 produces a chronic traumatic encephalopathy (CTE) protofilament conformation. Strikingly, one of the structures observed in the pS400 sample appeared to capture a secondary nucleation step. Together, these studies reveal the importance of positional effects of phosphorylation on tau self-assembly.

biochemistry↗

bioRxiv2026

Resolving the Activation Mechanism of the Human 20S Proteasome

Proteasome activators (PAs) bind the -subunits of the human 20S proteasome (h20S), opening the "gates" and allowing entry of substrate proteins. Aging is associated with diminished proteasome activity, leading to interest in understanding this activation mechanism. Evolving models have been proposed regarding PAs C-terminal tails, yet the critical molecular contacts for gate-opening are unclear. Here, we show a conserved leucine in the 5th position (P5) of the C-terminus is essential for h20S gate opening. By engineering C-termini in a model activator, PA26E102A, we show mutations to P5 systematically modulate proteasome activity in vitro and in cells. Structures of PA26E102A:h20S complexes at 2.7-3.2 [A] resolution identify interactions between P5 and a conserved arginine in the h20S, leading to partial or full gate opening. These results clarify the essential contacts required for h20S gate opening, potentially enabling design of proteasome activation therapies.

biophysics↗

bioRxiv2026

Structural Basis of Electron Transfer by the Human Nitric Oxide Synthase Holoenzyme Complex

Mammalian nitric oxide synthase (NOS) generates nitric oxide (NO), an essential signaling molecule in neurotransmission, inflammation, and cardiovascular regulation. NOS dysregulation contributes to neurodegeneration, septic shock, and ischemia. The structural basis of NOS function remains unclear, likely due to its dynamic, multi-component architecture. Catalysis requires electron transfer across five cofactors, with the oxygenase (Oxy) domain converting L-arginine to NO after receiving electrons from the reductase (Red) domain via calmodulin (CaM) activation. Here, we report cryo-electron microscopy structures of the human inducible NOS:CaM holoenzyme, revealing a previously unresolved architecture in which the Red:CaM arm spans the Oxy dimer in an apparent active state for cross-monomer electron transfer. The FMN subdomain is rotated [~]90{degrees} relative to the Red core, positioning its cofactor [~]9 [A] from the heme, adjacent to a conserved aromatic residue within a narrow Oxy domain tunnel. Red-Oxy flexibility in the active state may facilitate electron shuttling among cofactors, defining how NOS cofactors are transiently aligned to enable catalysis.

biochemistry↗

bioRxiv2026

Distinct tau filament folds in familial frontotemporal dementia due to the MAPT S305I mutation

Frontotemporal lobar degeneration with tau inclusions (FTLD-tau) comprise a class of fatal heterogeneous neurodegenerative diseases. Approximately 10% arise from pathogenic MAPT mutations and often cause severe, early-onset disease with pathology that is distinct yet partially overlapping with sporadic cases. Here, we evaluated post-mortem tissue from a patient with FTLD-tau due to MAPT S305I showing neuropathology most consistent with argyrophilic grain disease (AGD), a prevalent limbic tauopathy of aging. Structures determined by cryo-electron microscopy reveal tau filament folds that differ from those found in sporadic AGD or other tauopathies and feature a 4-layer architecture stabilized by the Ile substitution within its core. Comparative structural analysis reveals conserved motifs are shared among AGD, corticobasal degeneration, and MAPT P301T. A well-defined density stacks along a cationic cleft, indicative of a bound RNA-like polyanion or small-molecule. In vitro analysis shows the S305I mutation promotes fibrilization relative to normal tau. These results demonstrate that MAPT S305I stabilizes a distinct aggregation-prone tau fold that likely contributes to disease pathology and heterogeneity beyond its known splicing defects, and underscore potential limitations of using the most pathologically similar genetic form as a model for sporadic FTLD-tau.

neuroscience↗

bioRxiv2025

Structure-specific Mini-Prion Model for Alzheimer's Disease Tau Fibrils

A critical discovery of the past decade is that tau protein fibrils adopt disease-specific hallmark structures in each tauopathy. The faithful generation of synthetic fibrils adopting hallmark structures that can serve as targets for developing diagnostic and/or therapeutic strategies remains a grand challenge. We report on a rational design of synthetic fibrils built of a short peptide that adopts a critical structural motif in tauopathy fibrils found in Alzheimers Disease (AD) and Chronic Traumatic Encephalopathy (CTE). They serve as minimal prions with exquisite seeding competency, in vitro and in tau biosensor cells, for recruiting tau constructs ten times larger its size en route to AD or CTE fibril structures. We demonstrate that the generation of AD and CTE-like fibril structures is dramatically catalyzed in the presence of mini-AD prions and further influenced by salt composition in solution. Double Electron-Electron Resonance studies confirmed the preservation of AD-like folds across multi-generational seeding. Fibrils formed with the full AD/CTE-like core show strong seeding competency, with their templating effect dominating over the choice of salt composition that tunes the initial selection of AD- and CTE-like fibril populations. The mini-AD prions serve as a potent catalyst with templating capabilities that offer a novel strategy to design pathological tau fibril models.

biophysics↗

bioRxiv2025

Structures of Fab-stabilized CHIP reveal a conformational switch important in E3 ligase and chaperone functions

Carboxyl terminus of Hsc70-interacting protein (CHIP/STUB1) is a U-box E3 ligase essential for protein quality control, targeting misfolded or damaged proteins for clearance and conducting chaperone-like functions by suppressing aggregation of proteins, including tau. The previous structure of full-length CHIP identified an asymmetric homodimer in which one U-box is occluded from E2 binding, indicating an unusual half-of-sites activity. However, the flexibility of CHIP has complicated efforts to further characterize its structure and function. Here we leverage two CHIP-targeting fragment antigen-binding (Fab) antibodies to solve structures by cryo-EM. We identify one Fab binds to the CHIP U-box via interactions mimicking E2 contacts and stabilizes three distinct CHIP dimer states, revealing an asymmetric-to-symmetric conformational switch that would enable both U-box domains to be accessible for E2 binding. Conversely, the second Fab targets CHIPs coiled-coil domains, stabilizing the asymmetric dimer with a single accessible U-box. Remarkably, the Fabs exhibit opposing effects on CHIPs inhibition of tau aggregation, wherein binding to coiled-coil domains abolishes inhibition of aggregation, while binding to the U-box greatly potentiates this activity. Together, this work reveals how CHIP conformational states and binding interfaces may regulate ubiquitination cycles and chaperone-like functions.

biochemistry↗

bioRxiv2024

Structure of a novel α-synuclein filament fold from multiple system atrophy

Multiple system atrophy (MSA) is a synucleinopathy, a group of related diseases characterized by the accumulation of -synuclein aggregates in the brain. In MSA, these aggregates form glial cytoplasmic inclusions, which contain abundant cross-{beta} amyloid filaments. Structures of -synuclein filaments isolated from MSA patient tissue were determined by cryo-electron microscopy (cryo-EM), revealing three discrete folds that are distinct from -synuclein filaments associated with other synucleinopathies. Here, we use cryo-EM classification methods to characterize filaments from one individual with MSA and identify a novel, low-populated MSA filament fold (designated Type I2) in addition to a predominant class comprising MSA Type II2. The 3.3-[A] resolution structure of the Type I2 filament reveals a fold consisting of two asymmetric protofilaments. One is identical to a previously solved Type I protofilament, while the second adopts a novel fold that is chimeric between two previously reported Type I and II protofilaments. These results further define disease-specific folds of -synuclein filaments that develop in MSA and have implications for the design of therapeutic and diagnostic molecules that target disease.

biophysics↗

bioRxiv2024

Amyloid Accelerator Polyphosphate Implicated as the Mystery Density in α-Synuclein Fibrils

Aberrant aggregation of -Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived -Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates -Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in -Synuclein fibrils. Here we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the two critical lysine residues K43 and K45 leads to a loss of all previously reported effects of polyP binding on -Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo -Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.

biophysics↗

bioRxiv2024

Design principles to tailor Hsp104 therapeutics

The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated protein conformers. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can: (a) reduce Hsp70 collaboration without enhancing activity; (b) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s; (c) produce Hsp70-independent potentiated variants; or (d) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting the NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off- target toxicity, and counteract FUS proteinopathy in human cells. Thus, we establish important design principles to tailor Hsp104 therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/591398v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1520616org.highwire.dtl.DTLVardef@db3f5org.highwire.dtl.DTLVardef@1a1ae5eorg.highwire.dtl.DTLVardef@1beab7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Cryo-EM Structures Reveal Tau Filaments from Down Syndrome Adopt Alzheimer's Disease Fold

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimers disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-{beta} (A{beta}) precursor protein gene, the formation of self-propagating A{beta} and tau prion conformers, and the deposition of neurotoxic A{beta} plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36 to 63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-{beta}/{beta}-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

neuroscience↗

bioRxiv2023

Phosphorylation of a Cleaved Tau Proteoform at a Single Residue Inhibits Binding to the E3 Ubiquitin Ligase, CHIP

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimers disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to taus accumulation or what mechanisms might be involved. To explore these questions, we focused on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to block its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 revealed the mechanism of this clash and allowed design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We find that pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a discrete molecular mechanism for how phosphorylation at a specific site contributes to accumulation of an important tau proteoform.

biochemistry↗

bioRxiv2023

The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions

p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.

biochemistry↗

bioRxiv2023

Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly

The mitochondrial chaperonin, mtHsp60, promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin binds to clients and progresses through its ATP-dependent reaction cycle are not clear. Here, we determined cryo-electron microscopy (cryo-EM) structures of a hyperstable disease-associated mtHsp60 mutant, V72I, at three stages in this cycle. Unexpectedly, client density is identified in all states, revealing interactions with mtHsp60s apical domains and C-termini that coordinate client positioning in the folding chamber. We further identify a striking asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60/mtHsp10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify a new role for the apical domains in coordinating client capture and progression through the cycle, and suggest a conserved mechanism of group I chaperonin function.

biochemistry↗

bioRxiv2022

Stacked binding of a small molecule PET tracer to Alzheimer's tau paired helical filaments

Neurodegenerative diseases (NDs) are characterized by the formation of amyloid filaments that adopt disease-specific conformations in the brain. Recently developed small molecules hold promise as diagnostics and possible therapeutics for NDs, but their binding mechanisms to amyloid filaments remain unknown. Here, we used cryo-electron microscopy (cryo-EM) to determine a 2.7 [A] structure of Alzheimers disease patient-derived tau paired-helical filaments incubated with the GTP-1 PET probe. GTP-1 is bound stoichiometrically along an exposed cleft of each protofilament in a stacked arrangement that matches the fibrils symmetry. Multiscale modeling revealed favorable pi-pi aromatic stacking interactions between GTP-1 molecules that, together with small molecule-protein contacts, result in high affinity binding. This binding mode offers new insight into designing compounds for diagnosis and treatment of specific NDs. One Sentence SummaryCryo-EM structure reveals a novel stacked arrangement of the GTP-1 PET ligand bound to Alzheimers disease tau filaments.

biophysics↗

bioRxiv2022

The Chemical Features of Polyanions Modulate Tau Aggregation and Conformational States

The aggregation of tau into insoluble fibrils is a defining feature of neurodegenerative tauopathies. However, tau has a positive overall charge and is highly soluble; so polyanions, such as heparin, are typically required to promote its aggregation in vitro. There are dozens of polyanions in living systems and it is not clear which ones might promote this process. Here, we systematically measure the ability of 30 diverse, anionic biomolecules to initiate tau aggregation, using either wild type (WT) tau or the disease associated P301S mutant. We find that polyanions from many different structural classes can promote fibril formation and that P301S tau is sensitive to a greater number of polyanions (19/30) than WT tau (16/30). We also find that some polyanions preferentially reduce the lag time of the aggregation reactions, while others enhance the elongation rate, suggesting that they act on partially distinct steps. From the resulting structure-activity relationships, the valency of the polyanion seems to be an important chemical feature, such that anions with low valency tend to be weaker aggregation inducers, even at the same overall charge. Finally, the identity of the polyanion influences fibril morphology, based on electron microscopy and limited proteolysis. These results provide insight into the crucial role of polyanion--tau interactions in modulating tau conformational dynamics with implications for understanding the tau aggregation landscape in a complex cellular environment.

biochemistry↗

bioRxiv2022

Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with impaired protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). Yet how Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Unexpectedly, substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK-linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. Our findings illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.

biochemistry↗

bioRxiv2021

Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

The Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 [A] that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.

biophysics↗