Biology subjects

Griffin, P. R.

Publications and source records attributed to Griffin, P. R..

At least 19 recordsLinked to original sources

bioRxiv2026

PAR-Driven Condensation Maintains Stalled Replication Fork Stability

Poly(ADP-ribose) (PAR) is a nucleic acid-like heterogeneous polymer in nature. Recently, it was found to engage in liquid-liquid phase separation (LLPS), generating condensates as an emerging class of subcellular structures with pivotal functions in response to stimuli. As a post-translational modification catalyzed by PAR polymerases (PARPs), PAR is known to modulate many key events in cells. However, its involvement in biomolecular condensation remains elusive. Through an imaging-based screening of small molecules with diverse biological activities, we here discovered that PAR undergoes LLPS upon inhibiting proteasome in different types of cells, resulting in co-condensation of PAR with proteasome and ubiquitin chains in nucleus. This unprecedented co-condensation is dependent on PARP2 not PARP1 and requires K6-linked ubiquitylation. PAR is shown for the first time to directly interact with ubiquitin chains. Notably, stalled DNA replication forks arose from proteasome inhibition are co-localized with PAR-proteasome-ubiquitin chain condensates. By attenuating replication and stabilizing stalled replication forks, PAR-proteasome-ubiquitin chain condensates sustain genomic integrity under proteasomal stress. This work demonstrates a self-protective mechanism in stressed cells and provides fundamental understanding of PAR condensation in cell biology.

biochemistry↗

bioRxiv2025

Nucleosome Engagement Regulates RORγt Structure and Dynamics

ROR{gamma}t is a key transcriptional regulator that supports protective pathogen clearance but also drives autoimmune disorders. Genetic studies have identified ROR{gamma}t mutations that selectively protect from autoimmunity without compromising host defense. Herein, we describe the molecular principles governing how ROR{gamma}t selectively drives these context-dependent gene programs. Structural studies revealed the selectivity-determining mutations lie in an -helix, the C-terminal extension (CTE), which nucleates when ROR{gamma}t engages partially occluded DNA response elements at nucleosome entry/exit sites. In ex vivo Th17 cells, the CTE is required for full genomic occupancy and expression of inflammatory effector genes, while residual DNA binding supports partial occupancy that, alongside context-dependent transcription factors, induces lineage-stabilizing gene expression. These findings support a model where ROR{gamma}t uses distinct chromatin binding modes to engage an expansive chromatin landscape to activate target gene programs, explaining the complex phenotypes of CTE mutant mice.

biophysics↗

bioRxiv2025

A ternary switch determines ERα LBD conformation

The transcription factor estrogen receptor (ER) is the primary driver of ER+ breast cancer progression and a target of multiple FDA-approved anticancer drugs. Ligand-dependent activity of ER is determined by the conformation of helix-12 (H12) within the ligand binding domain (LBD), but how H12 transitions from an unliganded (apo) state to active (estrogen-bound) or inactive (SERM/SERD-bound) states remains unresolved. Here, we present the first crystal structure of an apo ER LBD, revealing a third distinct H12 conformation that regulates receptor activity. Structural mass-spectrometry, small-angle X-ray scattering, functional analysis and molecular dynamics simulations reveal that the apo conformation of H12 is stable in the absence of ligand, but is destabilised by Y537S and D538G breast cancer mutations driving constitutive activation. We propose a model in which H12 functions as a ternary molecular switch to determine receptor activity. These findings provide critical insights into the ligand-dependent and -independent regulation of ER and have significant implications for therapeutic intervention.

biochemistry↗

bioRxiv2024

Acetylation of RORβ by lysine acetyltransferase p300 and deacetylation by SIRT1 modulates receptor stability, turnover and transcriptional activity.

Retinoic acid receptor-related orphan receptor beta (ROR{beta}) is a transcription factor expressed in the central nervous system, retina, and bone that regulates circadian rhythms, retinal neurogenesis, and inflammatory signaling. Despite these critical functions, the mechanisms governing ROR{beta} stability remain poorly understood. Here, we identify a post-translational regulatory axis in which the lysine acetyltransferase p300 and the NAD-dependent deacetylase SIRT1 control ROR{beta} stability and transcriptional activity. p300-mediated acetylation increases ROR{beta} abundance, while SIRT1 modulates turnover through both catalytic and non-catalytic scaffolding mechanisms. K176 acetylation in the hinge primes UBC9/PIAS1-mediated SUMOylation at nearby K179, marking ROR{beta} for proteasomal degradation and reducing transcriptional output, providing a mechanistic framework for targeting ROR{beta} in neurological and retinal disorders, and bone homeostasis. HighlightsO_LIp300 acetylates ROR{beta} at eight lysines; SIRT1 reverses this via catalytic activity C_LIO_LIK176 acetylation primes UBC9/PIAS1-mediated SUMOylation at nearby K179 C_LIO_LISUMOylated ROR{beta} undergoes proteasomal degradation leading to reduced ROR{beta}-mediated transcriptional output C_LIO_LIp300 displaces ubiquitin E3 ligases from ROR{beta}; SIRT1 activation reduces PIAS1 association C_LI In BriefOLeary et al. define a post-translational circuit in which p300 acetylates K176 within the ROR{beta} hinge domain, priming SUMOylation at nearby K179 preferentially by UBC9/PIAS1. SUMOylated ROR{beta} is targeted for proteasomal degradation with diminished transcriptional activity, while SIRT1 deacetylase activity antagonizes this pathway via both catalytic activity and protein-protein interactions to stabilize active ROR{beta}. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/621067v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@16f18fforg.highwire.dtl.DTLVardef@d2208corg.highwire.dtl.DTLVardef@156d829org.highwire.dtl.DTLVardef@898e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Development of Clinically Viable Non-Muscle Myosin II Small Molecule Inhibitors with Broad Therapeutic Potential

Non-muscle myosin II (NMII), a molecular motor that regulates critical processes such as cytokinesis and neuronal synaptic plasticity, has substantial therapeutic potential. However, translating this potential to in vivo use has been hampered by the lack of selective tools. The most prototypical non-selective inhibitor, blebbistatin inactivates both NMII and cardiac myosin II (CMII), a key regulator of heart function. Using rational drug design, we developed a series of NMII inhibitors that improve tolerability by selectively targeting NMII over CMII, including MT-228, which has excellent properties such as high brain penetration and efficacy in preclinical models of stimulant use disorder, which has no current FDA-approved therapies. The structure of MT-228 bound to myosin II provides insight into its 17-fold selectivity for NMII over CMII. MT-228s broad therapeutic window opens the door to new disease treatments and provides valuable tools for the scientific community, along with promising leads for future medication development. HighlightsO_LIResearch suggests numerous indications, from axon regeneration and cancer, would benefit from a small molecule inhibitor of non-muscle myosin II, a molecular motor that regulates the actin cytoskeleton. C_LIO_LICurrent chemical probe options are very limited and lack sufficient safety for in vivo C_LIO_LIstudies, which we show is primarily due to potent inhibition of cardiac myosin II. C_LIO_LIRational design that focused on improving target selectivity over the pan-myosin II inhibitor, blebbistatin, led to the identification of MT-228, a small molecule inhibitor with a wide therapeutic window. C_LIO_LIHigh-resolution structure of MT-228 bound to myosin II reveals that selectivity results from a different positioning compared to blebbistatin and an important sequence difference between cardiac and non-muscle myosin II in the inhibitor binding pocket. C_LIO_LIA single administration of MT-228 shows long-lasting efficacy in animal models of stimulant use disorder, a current unmet and rapidly escalating need with no FDA-approved treatments. C_LI

cell biology↗

bioRxiv2024

MT-125 Inhibits Non-Muscle Myosin IIA and IIB, Synergizes with Oncogenic Kinase Inhibitors, and Prolongs Survival in Glioblastoma

We have identified a NMIIA and IIB-specific small molecule inhibitor, MT-125, and have studied its effects in GBM. MT-125 has high brain penetrance and retention and an excellent safety profile; blocks GBM invasion and cytokinesis, consistent with the known roles of NMII; and prolongs survival as a single agent in murine GBM models. MT-125 increases signaling along both the PDGFR- and MAPK-driven pathways through a mechanism that involves the upregulation of reactive oxygen species, and it synergizes with FDA-approved PDGFR and mTOR inhibitors in vitro. Combining MT-125 with sunitinib, a PDGFR inhibitor, or paxalisib, a combined PI3 Kinase/mTOR inhibitor significantly improves survival in orthotopic GBM models over either drug alone, and in the case of sunitinib, markedly prolongs survival in [~]40% of mice. Our results provide a powerful rationale for developing NMII targeting strategies to treat cancer and demonstrate that MT-125 has strong clinical potential for the treatment of GBM. HighlightsO_LIMT-125 is a highly specific small molecule inhibitor of non-muscle myosin IIA and IIB, is well-tolerated, and achieves therapeutic concentrations in the brain with systemic dosing. C_LIO_LITreating preclinical models of glioblastoma with MT-125 produces durable improvements in survival. C_LIO_LIMT-125 stimulates PDGFR- and MAPK-driven signaling in glioblastoma and increases dependency on these pathways. C_LIO_LICombining MT-125 with an FDA-approved PDGFR inhibitor in a mouse GBM model synergizes to improve median survival over either drug alone, and produces tumor free, prolonged survival in over 40% of mice. C_LI

cancer biology↗

bioRxiv2024

PPARG in osteocytes controls cell bioenergetics and systemic energy metabolism independently of sclerostin levels in circulation

ObjectiveThe skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels. MethodsIn vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp1CrePpar{gamma}flfl male and female mice ({gamma}OTKO) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Ppar{gamma}. As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. ResultsCirculating sclerostin levels of {gamma}OTKO male and female mice were not different from control mice. Male {gamma}OTKO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from {gamma}OTKO males suggested profound changes in cellular metabolism, fuel transport and usage, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, shifts in fuel utilization, and accumulation of reactive oxygen species (ROS). ConclusionsPPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism. HighlightsO_LIOsteocytes function as a body energostat via their bioenergetics C_LIO_LIPPARG protein acts as a "molecular break" of osteocyte mitochondrial activity C_LIO_LIPPARG deficiency activates TCA cycle, oxidative stress and ROS accumulation C_LIO_LIPPARG controls osteocyte insulin signaling and fuel utilization C_LI

physiology↗

bioRxiv2024

Structural insights into IMP2 dimerization and RNA binding

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to cancer tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state, with the KH12 domains of each IMP2 molecule forming the dimer interface. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function with more effective IMP2 inhibitors.

molecular biology↗

bioRxiv2022

Structural basis of interdomain communication in PPARγ

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates transcription via two activation function (AF) regulatory domains: a ligand-dependent AF-2 coregulator interaction surface within the C-terminal ligand-binding domain (LBD), and an N-terminal disordered AF-1 domain (NTD or A/B region) that functions through poorly understood structural mechanisms. Here, we show the PPAR{gamma} AF-1 contains an evolutionary conserved Trp-Pro motif that undergoes cis/trans isomerization, populating two long-lived conformations that participate in intradomain AF-1 and interdomain interactions including two surfaces in the C-terminal LBD ({beta}-sheet and the AF-2 surface), which are predicted in AlphaFold 3 models but not AlphaFold 2. NMR and chemical crosslinking mass spectrometry show that interdomain interactions occur for soluble isolated AF-1 and LBD proteins, as well as in full-length PPAR{gamma} in a phase separated state. Mutation of the region containing the Trp-Pro motif, which abrogates cis/trans isomerization of this region, impacts LBD interaction and reduces basal PPAR{gamma}-mediated transcription and agonist-dependent activation of PPAR{gamma}. Our findings provide structural insight into published in vitro and cellular studies that reported interdomain functional communication between the PPAR{gamma} AF-1 and LBD suggesting some of these effects may be mediated via AF-1/LBD interactions.

biochemistry↗

bioRxiv2022

Molecular glue CELMoD compounds are allosteric regulators of cereblon conformation

Cereblon (CRBN) is an ubiquitin ligase (E3) adaptor protein co-opted by CRBN E3 Ligase Modulatory Drugs (CELMoD) agents that target therapeutically-relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBNs Thalidomide Binding Domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We therefore performed cryo-EM analyses of the DDB1[~]CRBN apo-complex, and compared these structures with DDB1[~]CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an "open" conformation to the canonical "closed" conformation. Importantly, the neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy, and informing structure-guided design strategies to improve therapeutic efficacy.

molecular biology↗

bioRxiv2022

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its [~]30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate. TeaserWe elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.

biochemistry↗

bioRxiv2022

RORβ modulates a gene program that is protective against articular cartilage damage

Osteoarthritis (OA) is the most prevalent chronic joint disease which increases in frequency with age eventually impacting most people over the age of 65. OA is the leading cause of disability and impaired mobility, yet the pathogenesis of OA remains unclear. Treatments have focused mainly on pain relief and reducing joint swelling. Currently there are no effective treatments to slow the progression of the disease and to prevent irreversible loss of cartilage. Here we demonstrate that stable expression of ROR{beta} in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and is protective against development of OA. Specifically, we determined that ROR{beta} regulates the balance of FGFRs signaling on FGFR1/FGFR3 that ERK1/2-MAPK signaling was suppressed by FGFR1(cartilage destruction) and AKT signaling was enhanced by FGFR3 (cartilage protection). These results suggest a critical role for ROR{beta} in chondrogenesis and suggest that identification of mechanisms that control the expression of ROR{beta} in chondrocytes could lead to the development of disease modifying therapies for the treatment of OA.

cell biology↗

bioRxiv2021

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

The retinoic acid receptor-related orphan receptor {gamma} (ROR{gamma}) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of ROR{gamma} is far from complete. Like most nuclear receptors, ROR{gamma} must recruit coregulatory protein to enact the ROR{gamma} target gene program. To date, a majority of structural studies have been focused exclusively on the ROR{gamma} ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length ROR{gamma}:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that ROR{gamma} becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory proteins may be able to sense the ligand- and DNA-bound status of ROR{gamma}. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which ROR{gamma} is bound. Overall, the efforts described herein will illuminate important aspects of full length ROR{gamma} and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/445650v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f0a067org.highwire.dtl.DTLVardef@b2e9f6org.highwire.dtl.DTLVardef@1e15d5org.highwire.dtl.DTLVardef@12b8546_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

biophysics↗

bioRxiv2020

Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

molecular biology↗

bioRxiv2019

HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism1 to inflammation2 and cancer3. As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology4. However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor ROR{gamma} can be defined by selectively disrupting putative endogenous--but not synthetic--ligand binding. Furthermore, the characterization of a library of ROR{gamma} modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and site-directed mutagenesis to collectively reveal the structural determinants of ROR{gamma} ligand-dependent activation, critical for designing full agonists for application in cancer immunotherapy. Combined these observations support a model of receptor activation to more accurately describe ROR{gamma} pharmacology. Likewise, this bump-and-hole inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

biophysics↗

bioRxiv2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biophysics↗

bioRxiv2018

Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 [A] resolution, respectively. Furthermore, a 6.5 [A] reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

biochemistry↗