Open Conformation Research Articles

Closed and open structures of the eukaryotic magnesium channel Mrs2 reveal the auto-ligand-gating regulation mechanism.

The CorA/Mrs2 family of pentameric proteins are cardinal for the influx of Mg2+ across cellular membranes, importing the cation to mitochondria in eukaryotes. Yet, the conducting and regulation mechanisms of permeation remain elusive, particularly for the eukaryotic Mrs2 members. Here, we report closed and open Mrs2 cryo-electron microscopy structures, accompanied by functional characterization. Mg2+ flux is permitted by a narrow pore, gated by methionine and arginine residues in the closed state. Transition between the conformations is orchestrated by two pairs of conserved sensor-serving Mg2+-binding sites in the mitochondrial matrix lumen, located in between monomers. At lower levels of Mg2+, these ions are stripped, permitting an alternative, symmetrical shape, maintained by the RDLR motif that replaces one of the sensor site pairs in the open conformation. Thus, our findings collectively establish the molecular basis for selective Mg2+ influx of Mrs2 and an auto-ligand-gating regulation mechanism.

Constitutive opening of the Kv7.2 pore activation gate causes KCNQ2-developmental encephalopathy

Pathogenic variants in KCNQ2 encoding Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies (KCNQ2-encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro, and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S6 activation gate that undergoes large structural rearrangements during pore opening; the disease-causing A356T variant in KCNQ3, paralogous to the A317T variant in KCNQ2, was also investigated. Currents through KCNQ2 mutant channels displayed increased density, hyperpolarizing shifts in activation gating, faster activation and slower deactivation kinetics, and resistance to changes in the cellular concentrations of phosphatidylinositol 4,5-bisphosphate (PIP2), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in the probability of single-channel opening, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atom molecular dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, mutation-induced stabilization of the inner pore gate open configuration is a molecular pathogenetic mechanism for KCNQ2-related encephalopathies.

Open architecture of archaea MCM and dsDNA complexes resolved using monodispersed streptavidin affinity CryoEM

The cryo-electron microscopy (cryoEM) method has enabled high-resolution structure determination of numerous biomolecules and complexes. Nevertheless, cryoEM sample preparation of challenging proteins and complexes, especially those with low abundance or with preferential orientation, remains a major hurdle. We developed an affinity-grid method employing monodispersed single particle streptavidin on a lipid monolayer to enhance particle absorption on the grid surface and alleviate sample exposure to the air-water interface. Using this approach, we successfully enriched the Thermococcus kodakarensis mini-chromosome maintenance complex 3 (MCM3) on cryoEM grids through biotinylation and resolved its structure. We further utilized this affinity method to tether the biotin-tagged dsDNA to selectively enrich a stable MCM3-ATP-dsDNA complex for cryoEM structure determination. Intriguingly, both MCM3 apo and dsDNA bound structures exhibit left-handed open spiral conformations, distinct from other reported MCM structures. The large open gate is sufficient to accommodate a dsDNA which could potentially be melted. The value of mspSA affinity method was further demonstrated by mitigating the issue of preferential angular distribution of HIV-1 capsid protein hexamer and RNA polymerase II elongation complex from Saccharomyces cerevisiae.

Membrane lipid homeostasis dually regulates conformational transition of phosphoethanolamine transferase EptA

The phosphoethanolamine transferase EptA utilizes phosphatidylethanolamine (PE) in the bacterial cell membrane to modify the structure of lipopolysaccharide, thereby conferring antimicrobial resistance on Gram-negative pathogens. Previous studies have indicated that excessive consumption of PE can disrupt the cell membrane, leading to cell death. This implies the presence of a regulatory mechanism for EptA catalysis to maintain a balance between antimicrobial resistance and bacterial growth. Through microsecond-scale all-atom molecular dynamics simulations, we demonstrate that membrane lipid homeostasis modulates the conformational transition and catalytic activation of EptA. The conformation of EptA oscillates between closed and open states, ensuring the precise spatiotemporal sequence of substrates binding. Interestingly, the conformation of EptA is significantly influenced by its surrounding lipid microenvironment, particularly the PE proportion in the membrane. PE-rich membrane conditions initiate and stabilize the open conformation of EptA through both orthosteric and allosteric effects. Importantly, the reaction mediated by EptA gradually depletes PE in the membrane, ultimately hindering its conformational transition and catalytic activation. These findings collectively establish a self-promoted model, illustrating the regulatory mechanism of EptA during the development of antibiotic resistance.

Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for degradation.

Dominant negative mutations provide valuable tools for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative (DN) mutations in yeast Hsp90. In a previous mutational scan of the ATPase domain of Hsp90, we noticed that many mutations were at very low frequency after outgrowth in cells co-expressing WT Hsp90. Most of these depleted variants were located at the hinge of a lid that closes over ATP. To quantify toxic effects in the hinge regions, we performed mutational scanning using an inducible promoter and identified 113 variants with strong toxic effects. We analyzed individual DN mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. FRET assays performed on individual DN mutants indicate the linkage between ATPase activity and formation of the closed structure is disrupted. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. Biochemical analyses indicate that ATP hydrolysis by Hsp90 from open conformations can lead to ubiquitin-dependent client degradation.

Predicting multiple conformations of ligand binding sites in proteins suggests that AlphaFold2 may remember too much

The goal of this paper is predicting the conformational distributions of ligand binding sites using the AlphaFold2 (AF2) protein structure prediction program with stochastic subsampling of the multiple sequence alignment (MSA). We explored the opening of cryptic ligand binding sites in 16 proteins, where the closed and open conformations define the expected extreme points of the conformational variation. Due to the many structures of these proteins in the Protein Data Bank (PDB), we were able to study whether the distribution of X-ray structures affects the distribution of AF2 models. We have found that AF2 generates both a cluster of open and a cluster of closed models for proteins that have comparable numbers of open and closed structures in the PDB and not too many other conformations. This was observed even with default MSA parameters, thus without further subsampling. In contrast, with the exception of a single protein, AF2 did not yield multiple clusters of conformations for proteins that had imbalanced numbers of open and closed structures in the PDB, or had substantial numbers of other structures. Subsampling improved the results only for a single protein, but very shallow MSA led to incorrect structures. The ability of generating both open and closed conformations for six out of the 16 proteins agrees with the success rates of similar studies reported in the literature. However, we showed that this partial success is due to AF2 "remembering" the conformational distributions in the PDB and that the approach fails to predict rarely seen conformations.

Unraveling the Structural Basis of Biased Agonism in the β2-Adrenergic Receptor Through Molecular Dynamics Simulations.

Biased agonism in G protein-coupled receptors is a phenomenon resulting in the selective activation of distinct intracellular signaling pathways by different agonists, which may exhibit bias toward either Gs, Gi, or arrestin-mediated pathways. This study investigates the structural basis of ligand-induced biased agonism within the context of the β2-adrenergic receptor (β2-AR). Atomistic molecular dynamics simulations were conducted for β2-AR complexes with two stereoisomers of methoxynaphtyl fenoterol (MNFen), that is, compounds eliciting qualitatively different cellular responses. The simulations reveal distinct interaction patterns within the binding cavity, dependent on the stereoisomer. These changes propagate to the intracellular parts of the receptor, triggering various structural responses: the dynamic structure of the intracellular regions of the (R,R)-MNFen complex more closely resembles the "Gs-compatible" and "β-arrestin-compatible" conformation of β2-AR, while both stereoisomers maintain structural responses equidistant from the inactive conformation. These findings are confirmed by independent coarse-grained simulations. In the context of deciphered molecular mechanisms, Trp313 plays a pivotal role, altering its orientation upon interactions with (R,R)-MNFen, along with the Lys305-Asp192 ionic bridge. This effect, accompanied by ligand interactions with residues on TM2, increases the strength of interactions within the extracellular region and the binding cavity, resulting in a slightly more open conformation and a minor (by ca. 0.2 nm) increase in the distance between the TM5-TM7, TM1-TM6, TM6-TM7, and TM1-TM5 pairs. On the other hand, an even slighter decrease in the distance between the TM1-TM4 and TM2-TM4 pairs is observed.

Crystallographic Fragment Screening of a Bifunctional Proline Catabolic Enzyme Reveals New Inhibitor Templates for Proline Dehydrogenase and L-Glutamate-γ-semialdehyde Dehydrogenase.

The proline catabolic pathway consisting of proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde (GSAL) dehydrogenase (GSALDH) catalyzes the four-electron oxidation of L-proline to L-glutamate. Chemical probes to these enzymes are of interest for their role in cancer and inherited metabolic disease. Here, we report the results of a crystallographic fragment-screening campaign targeting both enzymes. A unique aspect of our approach is the screening of both enzymes simultaneously using crystals of the bifunctional PRODH-GSALDH enzyme, proline utilization A (PutA). A 288-fragment library from Zenobia was screened in crystallo in cocktails of six fragments. Validation X-ray crystallography with individual fragments identified seven crystal hits distributed in the PRODH active site, GSALDH aldehyde substrate-binding site, and GSALDH NAD+ adenine-binding site. The fragment bound in the PRODH active site, 4-methoxybenzyl alcohol, is structurally distinct from all known PRODH inhibitors as it lacks an anionic anchor and stabilizes open conformations of the active site, motivating the study of eighteen analogs. In total, thirteen crystal structures with resolutions ranging from 1.32 Å to 1.80 Å were determined, resolving the poses and interactions of seven fragments from the Zenobia library and five analogs of 4-methoxybenzyl alcohol. These results expand the chemical space of probes targeting proline catabolic enzymes and provide new structural information for further inhibitor development.

Inactivation of Pseudovirus Expressing the D614G Spike Protein Mutation using Nitric Oxide-Plasma Activated Water.

Variants of concern (VOCs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) exhibit high infectivity due to mutations, particularly in the spike protein, that facilitate enhanced binding of virus to human angiotensin-converting enzyme 2 (hACE2). The D614G mutation, situated in S1-domain, promotes the open conformation of spike protein, augmenting its interaction with hACE2. Activated water neutralizes pathogens by damaging biological molecules; however, its effect on mutated SARS-CoV-2 or VOCs requires further exploration. Here, the efficacy of nitric oxide (NOx)-plasma activated water (PAW) in inhibiting infections by SARS-CoV-2 pseudovirus expressing D614G-mutated spike protein is investigated, which serves as a model for mutated SARS-CoV-2. Results demonstrated high prevalence of D614G mutation in SARS-CoV-2 and its VOCs. NOx-PAW is non-toxic to cells at high concentration, inhibiting infection by 71%. Moreover, NOx-PAW induced structural changes in S1-domain of spike protein, reducing its binding affinity and lowering clathrin-mediated endocytosis-related gene expression. Additionally, in silico analysis revealed NOx species in NOx-PAW played key role in impairing S1-domain function of the mutated SARS-CoV-2 pseudovirus by interacting directly with it. Collectively, these findings reveal the potent inactivation ability of PAW against mutated SARS-CoV-2 and suggest its potential application in combating emerging variants of SARS-CoV-2 and other viral threats.

Inducible cell lines producing replication-defective human immunodeficiency virus particles containing envelope glycoproteins stabilized in a pretriggered conformation.

During the process by which human immunodeficiency virus (HIV-1) enters cells, the envelope glycoprotein (Env) trimer on the virion surface engages host cell receptors. Binding to the receptor CD4 induces Env to undergo transitions from a pretriggered, "closed" (State-1) conformation to more "open" (State 2/3) conformations. Most broadly neutralizing antibodies (bNAbs), which are difficult to elicit, recognize the pretriggered (State-1) conformation. More open Env conformations are recognized by poorly neutralizing antibodies (pNAbs), which are readily elicited during natural infection and vaccination with current Env immunogens. Env heterogeneity likely contributes to HIV-1 persistence by skewing antibody responses away from the pretriggered conformation. The conformationally flexible gp160 Env precursor on the infected cell or virion surface potentially presents multiple pNAb epitopes to the host immune system. Although proteolytic cleavage to produce the functional, mature Env trimer [(gp120/gp41)3] stabilizes State-1, many primary HIV-1 Envs spontaneously sample more open conformations. Here, we establish inducible cell lines that produce replication-defective HIV-1 particles with Env trimers stabilized in a pretriggered conformation. The mature Env is enriched on virus-like particles (VLPs). Using complementary approaches, we estimate an average of 25-50 Env trimers on each VLP. The stabilizing changes in Env limit the natural conformational heterogeneity of the VLP Env trimers, allowing recognition by bNAbs but not pNAbs. These defective VLPs provide a more homogeneous source of pretriggered Env trimers in a native membrane environment. Thus, these VLPs may facilitate the characterization of this functionally important Env conformation and its interaction with the immune system.IMPORTANCEA major impediment to the development of an effective HIV/AIDS vaccine is the inefficiency with which human immunodeficiency virus (HIV-1) envelope glycoproteins elicit antibodies that neutralize multiple virus strains. Neutralizing antibodies recognize a particular shape of the envelope glycoproteins that resides on the viral membrane before the virus engages the host cell. Here, we report the creation of stable cell lines that inducibly produce non-infectious HIV-like particles. The normally flexible envelope glycoprotein spikes on these virus-like particles have been stabilized in a conformation that is recognized by broadly neutralizing antibodies. These virus-like particles allow the study of the envelope glycoprotein conformation, its modification by sugars, and its ability to elicit desired neutralizing antibodies.

DISTANT RIBOSE 2'-O-METHYLATION OF 23S rRNA HELIX 69 PRE-ORDERS THE CAPREOMYCIN DRUG BINDING POCKET AT THE RIBOSOME SUBUNIT INTERFACE.

Loss of ribosomal RNA (rRNA) modifications incorporated by the intrinsic methyltransferase TlyA results in reduced sensitivity to tuberactinomycin antibiotics such as capreomycin. However, the mechanism by which rRNA methylation alters drug binding, particularly at the distant but functionally more important site in 23S rRNA Helix 69 (H69), is currently unknown. We determined high-resolution cryo-electron microscopy structures of the Mycolicibacterium smegmatis 70S ribosome with or without the two ribose 2'-O-methyl modifications incorporated by TlyA. In the unmodified ribosome, the tip of H69 adopts a more compact conformation, positioning two key nucleotides (A2137 and C2138) such that interactions with capreomycin would be lost and the binding pocket partially occluded. In contrast, methylation of 23S rRNA nucleotide C2144 results in conformational changes that propagate from the site of modification to the H69 tip, resulting in its movement away from h44, a more favorable positioning of C2138 and adoption of a more open conformation to enable capreomycin binding. Methylation of h44 also results in structural rearrangements at the H69-h44 interface that further support antibiotic binding. These structures thus reveal the effect and regulation of distant rRNA methylation on ribosome-targeting antibiotic binding.

Temperature and Ultrasound-Responsive Nanoassemblies for Enhanced Organ Targeting and Reduced Cardiac Toxicity.

Biocompatible nanocarriers are widely employed as drug-delivery vehicles for treatment. Nevertheless, indiscriminate drug release, insufficient organ-specific targeting, and systemic toxicity hamper nanocarrier effectiveness. Stimuli-responsive nano-sized drug delivery systems (DDS) are an important strategy for enhancing drug delivery efficiency and reducing unexpected drug release. This study introduces a temperature- and ultrasound-responsive nano-DDS in which the copolymer p-(MEO2MA-co-THPMA) is grafted onto mesoporous iron oxide nanoparticles (MIONs) to construct an MPL-p nano-DDS. The copolymer acts as a nanopore gatekeeper, assuming an open conformation at sub-physiological temperatures that allows drug encapsulation and a closed conformation at physiological temperatures that prevents unexpected drug release during circulation. Lactoferrin was conjugated to the nanoparticle surface via polyethylene glycol to gain organ-targeting ability. External ultrasonic irradiation of the nanoparticles in the targeted organs caused a conformational change of the copolymer and reopened the pores, facilitating controlled drug release. MPL-p exhibited excellent biocompatibility and rare drug release in circulation. When targeting delivery to the brain, ultrasound promoted the release of the loaded drugs in the brain without accumulation in other organs, avoiding the related adverse reactions, specifically those affecting the heart. This study established a novel temperature- and ultrasound-responsive DDS that reduced systemic adverse reactions compared with traditional DDS, especially in the heart, and demonstrated excellent organ delivery efficiency.

A convenient approach for generating dimeric nucleic acid dyes via click-chemistry

Fluorescent dyes are essential tools for visualizing DNA and RNA. Dimeric dyes like GelGreen have gained popularity as safer alternatives to ethidium bromide (EB) due to their reduced mutagenicity and genotoxicity. In this study, we present a straightforward method to synthesize novel acridine orange (AO)-based dimeric dyes using click chemistry. Starting from acridine orange, these dyes can be synthesized in just two steps. Compared to GelGreen, these new dyes incorporate additional triazole groups in their linkers. They exhibit a maximum absorption wavelength of approximately 472 nm, which shifts to around 503 nm upon binding with DNA, allowing excitation by blue light. These dyes show minimal fluorescence in aqueous solutions, indicating that they adopt a closed conformation where the fluorescence of acridine orange is quenched due to intramolecular aggregation. The presence of DNA significantly enhances their fluorescence at around 526 nm, suggesting that DNA binding induces an open conformation. This “light-up” property makes them highly sensitive DNA dyes with a strong signal-to-noise ratio. We successfully applied these novel dyes in agarose gel electrophoresis, where they demonstrated excellent performance.

Substrate-Based Ligand Design for Phenazine Biosynthesis Enzyme PhzF.

The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant "priority pathogens". In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3-hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3-hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors.

Mechanisms of Action Underlying Conductance-Modifying Positive Allosteric Modulators of the NMDA Receptor.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurologic, and neurodegenerative disorders. We recently described the thieno[2,3-day]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, human embryonic kidney cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 μM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances coagonist potency, and reduces single-channel conductance. We also show that EU1622-240 facilitates NMDAR activation when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg2+ sensitivity, in contrast to channels arising from activation of NMDARs with both coagonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. SIGNIFICANCE STATEMENT: NMDA receptors are expressed throughout the central nervous system and are permeable to calcium. EU1622-240 increases open probability and agonist potency while reducing single-channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one coagonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.

Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

Fusobacterium nucleatum is an oral commensal bacterium that can act as an opportunistic pathogen, and is implicated in diseases such as periodontitis, adverse pregnancy outcomes, colorectal cancer, and Alzheimer’s disease. F. nucleatum synthesizes lanthionine for its peptidoglycan, rather than meso-2,6-diaminopimelic acid (DAP) used by most Gram-negative bacteria. Despite lacking the biosynthetic pathway for DAP, the genome of F. nucleatum ATCC 25586 encodes a predicted DAP epimerase. A recent study hypothesized that this enzyme may act as a lanthionine epimerase, but the authors found a very low turnover rate, suggesting that this enzyme likely has another more favored substrate. Here, we characterize this enzyme as a histidine racemase (HisR), and found that catalytic turnover is ∼10,000× faster with L-histidine than with L,L-lanthionine. Kinetic experiments suggest that HisR functions as a cofactor-independent racemase and that turnover is specific for histidine, while crystal structures of catalytic cysteine to serine mutants (C67S or C209S) reveal this enzyme in its substrate-unbound, open conformation. Currently, the only other reported cofactor-independent histidine racemase is CntK from Staphylococcus aureus, which is used in the biosynthesis of staphylopine, a broad-spectrum metallophore that increases virulence of S. aureus. However, CntK shares only 28% sequence identity with HisR, and their genes exist in different genomic contexts. Knock-out of hisR in F. nucleatum results in a small but reproducible lag in growth compared to wild-type during exponential phase, suggesting that HisR may play a role in growth of this periodontal pathogen.

Opening and closing of a cryptic pocket in VP35 toggles it between two different RNA-binding modes.

Cryptic pockets are of growing interest as potential drug targets, particularly to control protein-nucleic acid interactions that often occur via flat surfaces. However, it remains unclear whether cryptic pockets contribute to protein function or if they are merely happenstantial features that can easily be evolved away to achieve drug resistance. Here, we explore whether a cryptic pocket in the Interferon Inhibitory Domain (IID) of viral protein 35 (VP35) of Zaire ebolavirus aids its ability to bind double-stranded RNA (dsRNA). We use simulations and experiments to study the relationship between cryptic pocket opening and dsRNA binding of the IIDs of two other filoviruses, Reston and Marburg. These homologs have nearly identical structures but block different interferon pathways due to different affinities for blunt ends and backbone of the dsRNA. Simulations and thiol-labeling experiments demonstrate that the homologs have varying probabilities of pocket opening. Subsequent dsRNA-binding assays suggest that closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone. Point mutations that modulate pocket opening proteins further confirm this preference. These results demonstrate the open cryptic pocket has a function, suggesting cryptic pockets are under selective pressure and may be difficult to evolve away to achieve drug resistance.

SMURF1 mediates damaged lysosomal homeostasis by ubiquitinating PPP3CB to promote the activation of TFEB

ABSTRACT The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.Abbreviation AID: autoinhibitory domain; ATG: autophagy related; CD: catalytic domain; CRD: carbohydrate-recognition domain; CsA: cyclosporin A; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complexes required for transport; GSK3B: glycogen synthase kinase 3 beta; LAMP1: lysosomal associated membrane protein 1; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NT: N-terminal tail; PPP3CB: protein phosphatase 3 catalytic subunit beta; PPP3R1: protein phosphatase 3 regulatory subunit B, alpha; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; VCP/p97: valosin containing protein; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.

Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells.

The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5-TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.

The discovery and structural basis of two distinct state-dependent inhibitors of BamA

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.