Biology subjects

Mohanty, P.

Publications and source records attributed to Mohanty, P..

16 recordsLinked to original sources

bioRxiv2026

Learning molecular determinants of selective small-molecule partitioning across biomolecular condensates

The functional role of biomolecular condensates is shaped by the composition of constituent proteins, nucleic acids, ions, and small molecules. Selective partitioning of small molecules into condensates has therefore emerged as a potential route to condensate-specific chemical probes and therapeutics. Although partitioning is influenced by differences in solvation environments between coexisting dense and dilute phases, a molecular framework connecting small-molecule structure to condensate-specific enrichment remains lacking. Here, we use existing experimental partitioning data for a library of FDA-approved drugs and metabolites across four biomolecular condensates to develop an interpretable graph-based model of small-molecule partitioning. By combining multitask pretraining, condensate-specific fine-tuning, evidential uncertainty quantification, and atom-level attribution analysis, our model predicts continuous partition coefficients with improved accuracy over descriptor-based approaches. Atom-level attributions reveal that condensate partitioning is not governed by a universal chemical rule: the same molecular scaffold can be read differently by distinct condensate environments, with local atomic context and connectivity determining whether specific atoms promote or suppress enrichment. We further apply the trained model to ~1.7 million drug-like molecules from ChEMBL, identifying a chemically diverse space of predicted condensate-selective partitioners and mapping regions where predictions are confident versus where new measurements would be most informative. Together, this work establishes condensate partitioning as a chemically learnable property shaped by the interplay between small-molecule structure and condensate-specific microenvironments, providing an interpretable and uncertainty-aware framework for defining molecular determinants of partitioning and guiding the discovery of condensate-selective small molecules.

biochemistry↗

bioRxiv2025

The Structural Basis for RNA Binding and Recognition of the Disordered Prion-Like Domain of TDP-43

Though the structural details of how RNA interacts with folded RNA-binding domains are well established, how intrinsically disordered regions (IDRs) found in a large fraction of RNA-binding proteins mediate contacts with RNA and if they contribute to binding specificity has not been extensively characterized. The human RNA-binding protein TDP-43 is associated with many RNA processing functions that require its predominantly disordered C-terminal domain (CTD) that forms disease-associated inclusions in ALS, and other neurodegenerative conditions. Here, we demonstrate that TDP-43 CTD directly interacts with RNA primarily via a region of the IDR composed of clustered positively charged residues. Large RNAs act as a multivalent scaffold for CTD monomers, inducing the -helical segment of TDP-43 CTD to form multimeric protein-protein structures. Additionally, we probe the nucleotide base and amino acid specificity of CTD-RNA interactions, showing that arginine, aromatic and polar residues display a preference for U and G nucleic acid bases over C and A. Finally, we probe the molecular basis for the strong binding interaction between TDP-43 and G4 quadruplex structures and discover similarly avid interactions with cytosine-rich DNA I-motifs. This work deepens our understanding of how disordered regions of proteins contribute to RNA recognition, drive function, and contribute to disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/695782v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f1d256org.highwire.dtl.DTLVardef@284c2forg.highwire.dtl.DTLVardef@1a1d715org.highwire.dtl.DTLVardef@2fbb1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Site-specific methionine oxidation alters structure and phase separation of TDP-43 C-terminal domain

TAR DNA binding protein 43 (TDP-43), a key protein linked to ALS pathology, undergoes phase separation and forms functional assemblies via condensation within cells. The conserved region (CR) within its C-terminal domain (CTD) mediates self-assembly through helix-helix interactions, while the flanking intrinsically disordered regions (IDRs) contribute to phase separation through transient interactions involving aromatic and hydrophobic residues. The CTD contains ten methionine residues distributed equally between these regions, making it particularly susceptible to oxidative modifications. While methionine oxidation is known to impair phase separation, neither the precise mechanism nor the specific contribution of methionines in the CR compared to the IDRs has been determined. Here, we combine NMR spectroscopy and all-atom molecular dynamics (MD) simulations to reveal if and how methionine oxidation in each region differentially affects CTD structure and phase separation. We demonstrate that all methionine residues are vulnerable to oxidation, leading to distinct regional effects: oxidation of CR methionines disrupts helical structure and directly impairs intermolecular helical association, while oxidation of IDR methionines disrupts long-range contacts. Hence, oxidation of methionines in both regions contributes to impaired phase separation, albeit through different mechanisms. These findings establish methionines as critical redox-sensitive modulators of TDP-43 phase behavior and provide molecular insights into how oxidative stress may contribute to TDP-43 dysregulation in neurodegenerative diseases.

biochemistry↗

bioRxiv2025

RNA modulates FUS condensate assembly, dynamics, and aggregation through diverse molecular contacts

Fused in sarcoma (FUS) is an RNA-binding protein that undergoes phase separation with RNA and other cellular components, forming ribonucleoprotein (RNP) granules. While recent advances delineating the molecular forces that underlie phase separation have largely focused on protein-protein interactions (1-6), the molecular details of protein-RNA interactions within condensates remain limited. In this study, we demonstrate that RNA modulates the phase separation of the low-complexity (LC) and arginine-glycine-glycine motif (RGG1) domains of FUS: low RNA concentrations enhance protein phase separation and excess RNA disrupts it. By integrating biochemical assays, NMR spectroscopy, and molecular dynamics simulations, we show that RNA incorporates into FUS condensates, reducing condensate density while enhancing local relaxation and diffusional motion of FUS. Surprisingly, whereas RNA binding in the dispersed phase primarily involves the RGG1 domain, within the condensed phase, both LC and RGG1 domains contribute to interactions with RNA. NMR and simulation data show diverse interactions between amino acids and RNA moieties, including prominent glutamine-RNA contacts, that stabilize FUS-RNA co-condensates. Furthermore, we found that RNA accelerates the liquid-to-solid transition of FUS LC-RGG1 condensates, promoting fibrillar aggregate formation. Together, these results provide mechanistic insight into how RNA regulates the assembly, dynamics, and maturation of protein condensates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/694118v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@19168a9org.highwire.dtl.DTLVardef@16e6878org.highwire.dtl.DTLVardef@18cb8a9org.highwire.dtl.DTLVardef@1af6a4c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Using NMR and molecular simulations, we map how RNA engages FUS LC-RGG1 within condensates through electrostatic, {pi}-stacking, and hydrogen-bond contacts. We find that RNA incorporation dilutes condensate density, tunes protein mobility, remodels interaction networks, and accelerates the formation of fibrillar aggregates. C_FIG

biochemistry↗

bioRxiv2025

Multiscale simulations elucidate the mechanism of polyglutamine aggregation and the role of flanking domains in fibril polymorphism

Protein aggregation, which is implicated in aging and neurodegenerative diseases, typically involves a transition from soluble monomers and oligomers to insoluble fibrils. Polyglutamine (polyQ) tracts in proteins can form amyloid fibrils, which are linked to polyQ diseases, including Huntingtons disease (HD), where the length of the polyQ tract inversely correlates with the age of onset. Despite significant research on the mechanisms of Httex1 aggregation, atomistic information regarding the intermediate stages of its fibrillation and the morphological characteristics of the end-state amyloid fibrils remains limited. Recently, molecular dynamics (MD) simulations based on a hybrid multistate structure-based model, Multi-eGO, have shown promise in capturing the kinetics and mechanism of amyloid fibrillation with high computational efficiency while achieving qualitative agreement with experiments. Here, we utilize the multi-eGO simulation methodology to study the mechanism and kinetics of polyQ fibrillation and the effect of the N17 flanking domain of Huntingtin protein. Aggregation simulations of polyQ produced highly heterogeneous amyloid fibrils with variable-width branched morphologies by incorporating combinations of {beta}-turn, {beta}-arc, and {beta}-strand structures, while the presence of the N17 flanking domain reduces amyloid fibril heterogeneity by favoring {beta}-strand conformations. Our simulations reveal that the presence of N17 domain enhanced aggregation kinetics by promoting the formation of large, structurally stable oligomers. Furthermore, the early-stage aggregation process involves two distinct mechanisms: backbone interactions driving {beta}-sheet formation and side-chain interdigitation. Overall, our study provides detailed insights into fibrillation kinetics, mechanisms, and end-state polymorphism associated with Httex1 amyloid aggregation. SIGNIFICANCE STATEMENTPolyglutamine (polyQ) aggregation is central to Huntingtons disease and related neurodegenerative disorders. Despite extensive experimental efforts, a complete molecular understanding of this process--from early aggregation events to the origins of fibril polymorphism--has remained elusive, with varied interpretations of complex fibril architectures. Through multiscale simulations, we reveal how polyQ fibrils adopt diverse tertiary and quaternary structures and demonstrate how the N-terminal flanking domain (N17) modulates fibril architecture and accelerates aggregation. Our hybrid multi-eGO simulations capture early-stage fibrillation kinetics and identify distinct structural polymorphs that align with experimental observations. This work provides a molecular framework for understanding amyloid polymorphism and illuminates the role of flanking domains in shaping aggregation pathways-- offering valuable insights for therapeutic strategies targeting early toxic intermediates.

biophysics↗

bioRxiv2025

Elucidation of the molecular interaction network underlying full-length FUS conformational transitions and its phase separation using atomistic simulations

Fused in Sarcoma (FUS), a multi-domain RNA-binding protein, orchestrates cellular functions through liquid-liquid phase separation (LLPS), which promotes the formation of biomolecular condensates in vivo. While crucial to understanding cellular processes, an atomic-level view of the interdomain interactions associated with full-length (FL) FUS LLPS remains challenging due to its low solubility in vitro. Here, using all-atom (AA) molecular dynamics (MD) simulations, we examined the conformational dynamics and interdomain interactions of FL FUS in both dilute and condensed phases. Comparing two modern force fields (FFs) - Amber ff03ws and ff99SBws-STQ, we found that monomer simulation ensembles generated by both FFs exhibited qualitatively similar intramolecular interaction profiles dominated by intrinsically disordered regions (IDRs). While the two folded domains minimally participated in interdomain interactions, their stabilities significantly influenced the chain dimension and led to discrepancies compared to experimental data for both FFs. We observed that the Amber ff99SBws-STQ coupled with bond parameters adopted from the Zinc Amber force field (ZAFF) maintained stable folded domains and improved estimates of the chain dimensions. Finally, a microsecond-timescale simulation of FL FUS condensate revealed an extensive network of electrostatic interactions which are strongly correlated with those that modulate the dilute phase chain dimensions. Overall, insights from our all-atom simulations illuminate the interplay between folded domain stability and IDR interactions in modulating protein conformation and phase separation, advancing our understanding of FUS-related pathologies at the molecular level and aiding in the development of new therapeutics.

biophysics↗

bioRxiv2024

A genome-wide genetic screen identified targets for destabilizing the parasitophorous vacuole of Chlamydia trachomatis

The bacterial pathogen Chlamydia trachomatis employs the effector CpoS to suppress a host defense response that aborts intracellular bacterial growth by inducing host cell death. While conducting a CRISPR knock-out screen for genes contributing to this response, we uncovered a mutant deficient for CpoS to display a markedly increased reliance on host cellular ceramide synthesis, compensating for its diminished ability to acquire sphingolipids via modulating membrane trafficking. Employing the power of the just recently established molecular genetic toolbox for Chlamydia, we developed an innovative microscopic reporter system that revealed the mutant to thrive in unstable parasitophorous vacuoles (inclusions), characterized initially by the release of individual bacteria from otherwise intact-appearing vacuoles. CpoS-deficient inclusions were further destabilized by disruptions in ceramide synthesis, while supplementation of sphingoid bases stabilized them, also preventing the defensive host cell death response. Notably, early inclusion destabilization, achieved by simultaneous disruption of two transport routes, caused infection clearance without damaging the host cells. Overall, this study highlights the inclusions role as a refuge, demonstrates CpoS to maintain inclusion integrity by ensuring sphingolipid supply, and provides directions for a future therapeutic exploitation. SIGNIFICANCEA wide range of clinically significant microbes evolved to hide from the intrinsic defenses of their host cells by thriving within membrane-enclosed pathogen-containing vacuoles. This raises the intriguing possibility that such vacuoles could be targeted therapeutically. The bacterial pathogen Chlamydia trachomatis could be an exceptionally well-suited target for such innovative medicines given its medical importance and strict dependence on host cells. However, progress has been stalled by the lack of sensitive tools for detecting inclusion damage. Here, we resolved this major technical roadblock and uncovered the pathogen to employ the secreted effector CpoS, a modulator of membrane trafficking, to stabilize its vacuole by ensuring adequate sphingolipid supply. These methodological advances and mechanistic insights should promote the development of vacuole-destabilizing therapeutics.

microbiology↗

bioRxiv2024

Structural details of helix-mediated TDP-43 C-terminal domain multimerization

The primarily disordered C-terminal domain (CTD) of TAR DNA binding protein-43 (TDP-43), a key nuclear protein in RNA metabolism, forms neuronal inclusions in several neurodegenerative diseases. A conserved region (CR, spanning residues 319-341) in CTD forms transient helix-helix contacts important for its higher-order oligomerization and function that are disrupted by ALS-associated mutations. However, the structural details of CR assembly and the explanation for several ALS-associated variants impact on phase separation and function remain unclear due to challenges in analyzing the dynamic association of TDP-43 CTD using traditional structural biology approaches. By employing an integrative approach, combining biophysical experiments, biochemical assays, AlphaFold2-Multimer (AF2-Multimer), and atomistic simulations, we generated structural models of helical oligomerization of TDP-43 CR. Using NMR, we first established that the native state of TDP-43 CR under physiological conditions is -helical. Next, alanine scanning mutagenesis revealed that while hydrophobic residues in the CR are important for CR assembly, phase separation and TDP-43 nuclear retention function, polar residues down regulate these processes. Finally, pairing AF2-Multimer modeling with AAMD simulations indicated that dynamic, oligomeric assemblies of TDP-43 that are stabilized by a methionine-rich core with specific contributions from a tryptophan/leucine pair. In conclusion, our results advance the structural understanding of the mechanisms driving TDP-43 function and provide a window into the initial stages of its conversion into pathogenic aggregates.

biochemistry↗

bioRxiv2024

Transient interdomain interactions modulate the monomeric structural ensemble and oligomerization landscape of Huntingtin Exon 1

Polyglutamine expansion ([≥] 36 residues) within the N-terminal exon-1 of Huntingtin (Httex1) leads to Huntingtons disease, a neurodegenerative condition marked by the presence of intranuclear Htt inclusions. Notably, the polyglutamine tract in Httex1 is flanked by an N-terminal coiled-coil domain - N17 (17 amino acids), which undergoes self-association to promote the formation of soluble Httex1 oligomers and brings the aggregation-prone polyQ tracts in close spatial proximity. However, the mechanisms underlying the subsequent conversion of soluble oligomers into insoluble {beta}-rich aggregates with increasing polyQ length, remain unclear. Current knowledge suggests that expansion of the polyQ tract increases its helicity, and this favors its oligomerization and aggregation. In addition, studies utilizing photocrosslinking, conformation-specific antibodies and a stable coiled-coil heterotetrametric system fused to polyQ indicate that domain "cross-talk" (i.e., interdomain interactions) may play a role in the emergence of toxic conformations and the conversion of Httex1 oligomers into fibrillar aggregates. Here, we performed extensive atomistic molecular dynamics (MD) simulations (aggregate time [~] 0.7 ms) to uncover the interplay between structural transformation and domain "cross-talk" on the conformational ensemble and oligomerization landscape of Httex1. Notably, our MD-derived ensembles of N17-polyQ monomers validated against 13C NMR chemical shifts indicated that in addition to elevated - helicity, polyQ expansion also favors transient, interdomain (N17-polyQ) interactions which result in the emergence of {beta}-sheet conformations. Further, interdomain interactions competed with increased polyQ tract -helicity to modulate the stability of N17-mediated dimers and thereby promoted a heterogenous dimerization landscape. Finally, we observed that the C-terminal proline-rich domain (PRD) promoted condensation of Httex1 through self-interactions involving its P10/P11 tracts while also interacting with N17 to suppress its -helicity. In summary, our study demonstrates a significant role for domain "cross-talk" in modulating the monomeric structural ensemble and self-assembly of Httex1.

biophysics↗

bioRxiv2024

RNA-binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs

TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 towards its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa:104-263). Importantly, the loss of RNA-binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA in TDP-43 function and pathology, a comprehensive characterization of the effect of RNA-binding on conformational dynamics, interdomain interactions and intradomain stability of the tandem RRMs has not yet been conducted. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA-binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. Our simulations reveal a high intrinsic conformational plasticity of the tandem RRMs in the absence of RNA which surprisingly, is accompanied by a tendency of RRM1 to adopt partially-unfolded conformations. While binding to RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability, several RRM-RNA contacts mediated by highly conserved residues are observed to be far more dynamic than previously inferred from NMR structural ensemble. Overall, our simulations reveal how RNA dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation. SIGNIFICANCEThe cytoplasmic mislocalization and aggregation of TDP-43 due to loss of its RNA-binding capability is associated with the onset and progression of neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Due to the flexible nature of RNA and the presence of a disordered linker between RRM domains, characterizing the dynamic interactions between RRMs-RNA and/or RRM1-RRM2 by experiments alone has remained challenging. In this study, we performed all-atom simulations initiated from the NMR conformers of RNA-bound tandem RRMs of TDP-43 to investigate their underlying structural and conformational dynamics. Our findings indicate that RNA binding effectively reduces conformational heterogeneity in the tandem RRMs and acts as a protective factor for the unfolding and aggregation of RRM1. These effects are achieved through a combination of stable and dynamic protein-RNA interactions which involve highly conserved amino acids.

biophysics↗

bioRxiv2024

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

cell biology↗

bioRxiv2023

Bacterial deamidases modulate ubiquitin structure and dynamics to dysregulate ubiquitin signaling

The deamidases secreted by Burkholderia pseudomallei and Enteropathogenic Ecoli modify the residue 40 in ubiquitin from a Glutamine (Q) to Glutamate (E), triggering several downstream processes to cause cell cycle arrest and activate immune responses. Deamidation hampers the activity of ubiquitin and its interaction with ubiquitin chain receptors by an unknown mechanism. Here, we study the effect of deamidation on ubiquitin structure and dynamics. We report the crystal structure of the deamidated ubiquitin, supported by NMR and molecular dynamics simulations. The structure reveals a new intra-molecular salt bridge between the deamidated region and the C-terminal tail of ubiquitin. The salt bridge perturbs the dynamics of the ubiquitin tail to reduce affinity for ubiquitin receptors like the p62 ubiquitin-associated domain. The salt bridge disrupts the transition to catalytically active E2~Ub closed conformation. Consequently, RING E3s fail to interact with E2~Ub, reducing ubiquitination activity. Our findings reveal that deamidation-induced intramolecular salt bridges in ubiquitin modulate conformational ensembles to inactivate ubiquitination.

biochemistry↗

bioRxiv2022

Aliphatic residues contribute significantly to the phase separation of TDP-43 C-terminal domain

TAR DNA binding protein 43 (TDP-43) is involved in key processes in RNA metabolism such as splicing, stability and transcription. TDP-43 dysfunction is frequently implicated in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). The prion-like, disordered C-terminal domain (CTD) of TDP-43 is aggregation-prone and harbors the majority (~90%) of all ALS-related mutations. Recent studies have established that TDP-43 CTD can undergo liquid-liquid phase separation (LLPS) in isolation and is important for phase separation (PS) of the full-length protein under physiological conditions. While a short conserved helical region (CR, spanning residues 319-341) promotes oligomerization and is essential for LLPS, aromatic residues in the flanking disordered regions (IDR1/2) have also been found to play a critical role in PS and aggregation. However, TDP-43 CTD has a distinct sequence composition compared with other phase separating proteins, including many aliphatic residues. These residues have been suggested to modulate the apparent viscosity of the resulting phases, but their direct contribution to phase separation has been relatively ignored. Here, we utilized a multiscale simulation and experimental approach to assess the residue-level determinants of TDP-43 CTD phase separation. Single chain and condensed phase simulations performed at the atomistic and coarse-grained level respectively, identified the importance of aromatic residues (previously established) while also suggesting an essential role for aliphatic methionine residues in LLPS. In vitro experiments confirmed the role of phenylalanine, methionine, and leucine (but not alanine) residues in driving the phase separation of CTD, which have not been previously considered essential for describing the molecular grammar of PS. Finally, NMR experiments also showed that phenylalanine residues in the disordered flanking regions and methionine residues both within and outside the CR contribute important contacts to CTD interactions. Broadly, our work highlights the importance of non-alanine aliphatic residues such as methionine and leucine, and potentially valine and isoleucine, in determining the LLPS propensity, expanding the molecular grammar of protein phase separation to include critical contributions from aliphatic residues.

biophysics↗

bioRxiv2022

Zn-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

The toxic gain of function of Cu/Zn superoxide dismutase (SOD1) associated with the neurodegenerative disease - Amyotrophic lateral sclerosis (ALS), is believed to occur via misfolding and/or aggregation. SOD1 is also associated with stress granules (SGs) which are a type of membraneless organelle believed to form via liquid-liquid phase separation (LLPS) of several proteins containing low-complexity, disordered regions. Using a combination of experiments and computer simulations, we report here that structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The phase-separated droplets give rise to aggregates which eventually form toxic amyloids upon prolonged incubation. The addition of exogenous Zn to immature, metal-free SOD1 and the severe ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. In contrast, the Zn-induced inhibition of LLPS and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In conclusion, our work establishes a role for Zn-dependent modulation of SOD1 disorder and LLPS as a precursor phenomenon which may lead to the formation of toxic amyloids associated with ALS. Significance StatementThe formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. SOD1 is a metalloenzyme which is stabilized by the metal co-factor - Zn. In this work, we utilize experimental and simulation techniques to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.

biophysics↗

bioRxiv2021

Functional insights of two MATE transporters from Vibrio fluvialis

Functional characterization of H- and D-MATE (Multidrug and Toxin Extrusion) transporters from clinical isolates of Vibrio fluvialis revealed H-type conferred resistance to fluoroquinolones, ethidium bromide and safranin whereas D-type exhibited marginal resistance towards ethidium bromide only. Both H-/D-type transporters were inhibited by reserpine resulting in increased intracellular norfloxacin concentration. The efflux was facilitated by both Na+/K+ ions, suggesting that these efflux pumps were ion-dependent. In presence of various classes of EPIs, there was decrease in MIC exhibited by H-/D-type efflux pumps towards norfloxacin which didnt translate into transport inhibition. But reserpine presented a conclusive pattern with decrease in MIC towards norfloxacin and increased norfloxacin accumulation inferring maximum inhibition. Substrate binding and electrostatic charge distribution of both the transporters was similar to other known MATE transporters. The H-type exhibited 10 transmembranes and D-type exhibited 11 TMs which was different from other MATE transporters known to have 12 TMs (Transmembranes). Data derived from molecular docking and ion binding studies revealed that Aspartic Acid residue in 1st TM acts as ion binding site with transport mechanism similar to NorM. Electrostatic potential map of both the transporters revealed that there is a cavity formation within the transporters surrounded by charged electronegative amino acid residues. Interestingly, surface models of both transporters revealed that 1st TM forms covalent bond with 7th TM towards extracellular space. Docking studies also revealed that reserpine covalently binds to central pocket of both transporters and serves as excellent EPI against these transporters as evidenced by MIC and drug accumulation assays.

microbiology↗

bioRxiv2019

Deamidation disrupts native and transient contacts to weaken the interaction between UBC13 and RING-finger E3 ligases

The deamidase OspI from enteric bacteria Shigella flexneri deamidates a glutamine residue in the host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate (Q100E). Consequently, its polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream NF-{kappa}B inflammatory response is inactivated. The precise role of deamidation in inactivating the UBC13/TRAF6 complex is unknown. We report that deamidation inhibits the interaction between UBC13 and TRAF6 RING-domain (TRAF6RING) by perturbing both the native and transient interactions. Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a critical intermolecular salt-bridge at the native UBC13/TRAF6RING interface. Moreover, the salt-bridge competition prevents transient interactions necessary to form a typical UBC13/RING complex. Repulsion between E100 and the negatively charged surface of RING also prevents transient interactions in the UBC13/RING complex. Our findings highlight a mechanism where a post-translational modification perturbs the conformation and stability of transient complexes to inhibit protein-protein association.

biochemistry↗