Biology subjects

Mohanty, P.

Publications and source records attributed to Mohanty, P..

5 recordsLinked to original sources

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↗